Evaluation of a global spring wheat panel for stripe rust: Resistance loci validation and novel resources identification.

Stripe rust (incited by Puccinia striiformis f. sp. tritici) is airborne wheat (Triticum aestivum L.) disease with dynamic virulence evolution. Thus, anticipatory and continued screening in hotspot regions is crucial to identify new pathotypes and integrate new resistance resources to prevent potential disease epidemics. A global wheat panel consisting of 882 landraces and 912 improved accessions was evaluated in two locations in Egypt during 2016 and 2017. Five prevalent and aggressive pathotypes of stripe rust were used to inoculate the accessions during the two growing seasons and two locations under field conditions. The objectives were to evaluate the panel for stripe rust resistance at the adult plant stage, identify potentially novel QTLs associated with stripe rust resistance, and validate previously reported stripe rust QTLs under the Egyptian conditions. The results indicated that 42 landraces and 140 improved accessions were resistant to stripe rust. Moreover, 24 SNPs were associated with stripe rust resistance and were within 18 wheat functional genes. Four of these genes were involved in several plant defense mechanisms. The number of favorable alleles, based upon the associated SNPs, was significant and negatively correlated with stripe rust resistance score, i.e., as the number of resistances alleles increased the observed resistance increased. In conclusion, generating new stripe rust phenotypic information on this panel while using the publicly available molecular marker data, contributed to identifying potentially novel QTLs associated with stripe rust and validated 17 of the previously reported QTLs in one of the global hotspots for stripe rust.

Populations of doubled haploids for genetic mapping in hexaploid winter triticale.

To create a framework for genetic dissection of hexaploid triticale, six populations of doubled haploid (DH) lines were developed from pairwise hybrids of high-yielding winter triticale cultivars. The six populations comprise between 97 and 231 genotyped DH lines each, totaling 957 DH lines. A consensus genetic map spans 4593.9 cM is composed of 1576 unique DArT markers. The maps reveal several structural rearrangements in triticale genomes. In preliminary tests of the populations and maps, markers specific to wheat segments of the engineered rye chromosome 1R (RM1B) were identified. Example QTL mapping of days to heading in cv. Krakowiak revealed loci on chromosomes 2BL and 2R responsible for extended vernalization requirement, and candidate genes were identified. The material is available to all parties interested in triticale genetics.

A comparison between genotyping-by-sequencing and array-based scoring of SNPs for genomic prediction accuracy in winter wheat.

The utilization of DNA molecular markers in plant breeding to maximize selection response via marker-assisted selection (MAS) and genomic selection (GS) has revolutionized plant breeding. A key factor affecting GS applicability is the choice of molecular marker platform. Genotyping-by-sequencing scored SNPs (GBS-scored SNPs) provides a large number of markers, albeit with high rates of missing data. Array scored SNPs are of high quality, but the cost per sample is substantially higher. The objectives of this study were 1) compare GBS-scored SNPs, and array scored SNPs for genomic selection applications, and 2) compare estimates of genomic kinship and population structure calculated using the two marker platforms. SNPs were compared in a diversity panel consisting of 299 hard winter wheat (Triticum aestivum L.) accessions that were part of a multi-year, multi-environments association mapping study. The panel was phenotyped in Ithaca, Nebraska for heading date, plant height, days to physiological maturity and grain yield in 2012 and 2013. The panel was genotyped using GBS-scored SNPs, and array scored SNPs. Results indicate that GBS-scored SNPs is comparable to or better than Array-scored SNPs for genomic prediction application. Both platforms identified the same genetic patterns in the panel where 90% of the lines were classified to common genetic groups. Overall, we concluded that GBS-scored SNPs have the potential to be the marker platform of choice for genetic diversity and genomic selection in winter wheat.

Identification of markers linked to genes for sprouting tolerance (independent of grain color) in hard white winter wheat (HWWW).

KEY MESSAGE: Hard red wheats can donate genes to hard white wheats for tolerance to preharvest sprouting, the effects are quantitative in nature, and may be tracked with previously described DNA markers. ABSTRACT: Pre-harvest sprouting (PHS) of wheat (Triticum aestivum L.) can negatively impact end-use quality and seed viability at planting. Due to preferences for white over red wheat in international markets, white wheat with PHS tolerance has become increasingly desired for worldwide wheat production. In general, however, red wheat is more tolerant of sprouting than white wheat. The main objective of this study was the identification of PHS tolerance conditioned by genes donated from hard red winter wheat, using markers applicable to the Great Plains hard white wheat gene pool. Three red wheat by white wheat populations, Niobrara/NW99L7068, NE98466/NW99L7068 and Jagalene/NW99L7068 were developed, and white-seeded progenies were analyzed for PHS tolerance and used to identify markers for the trait. In the three populations, marker loci with significant allelic effects were most commonly located on chromosomes of group 2, 3, 4 and 5, though additional markers were detected across the wheat genome. Chromosome 3A was the only chromosome with significant markers in all three populations. Markers were inconsistent across the three populations, and markers linked to tolerance-inducing loci were identified in both tolerant and susceptible parents. Additive effects of marker loci were common. In the present investigation, a wide range of PHS tolerance was observed, even though all lines were fixed for the recently reported positive TaPHS1 allele. PHS tolerance is controlled by additive major gene effects with minor gene effects where variations of minor gene effects were still unclear.

Quantification of Yield Loss Caused by Triticum mosaic virus and Wheat streak mosaic virus in Winter Wheat Under Field Conditions.

Triticum mosaic virus (TriMV) and Wheat streak mosaic virus (WSMV) infect winter wheat (Triticum aestivum) in the Great Plains region of the United States. The two viruses are transmitted by wheat curl mites (Aceria tosichella), which also transmit High Plains virus. In a field study conducted in 2011 and 2012, winter wheat cultivars Millennium (WSMV-susceptible) and Mace (WSMV-resistant) were mechanically inoculated with TriMV, WSMV, TriMV+WSMV, or sterile water at the two-leaf growth stage. Chlorophyll meter (soil plant analysis development [SPAD]) readings, area under the SPAD progress curve (AUSPC), grain yield (=yield), yield components (spikes/m2, kernels/spike, 1,000-kernel weight), and aerial dry matter were determined. In Millennium, all measured variables were significantly reduced by single or double virus inoculation, with the greatest reductions occurring in the double-inoculated treatment. Among the yield components, the greatest reductions occurred in spikes/m2. In Mace, only AUSPC was significantly reduced by the TriMV+WSMV treatment in 2012. There was a significant (P ≤ 0.05), negative linear relationship between SPAD readings and day of year in all inoculation treatments in Millennium and in the TriMV+WSMV treatment in Mace. There were significant (P ≤ 0.05), positive linear relationships between yield and SPAD readings and between yield and aerial dry matter in Millennium but not in Mace. The results from this study indicate that under field conditions, (i) Mace, a WSMV-resistant cultivar, is also resistant to TriMV, and (ii) double inoculation of winter wheat by TriMV and WSMV exacerbates symptom expression and yield loss in a susceptible cultivar.

Inheritance of grain polyphenol oxidase (PPO) activity in multiple wheat (Triticum aestivum L.) genetic backgrounds.

Grain polyphenol oxidase (PPO) activity can cause discoloration of wheat (Triticum aestivum L.) food products. Five crosses (PI 117635/Antelope; Fielder/NW03681; Fielder/Antelope; NW07OR1070/Antelope; NW07OR1066/OR2050272H) were selected to study the genetic inheritance of PPO activity. STS markers, PPO18, PPO29 and STS01, were used to identify lines with putative alleles at the Ppo-A1 and Ppo-D1 loci conditioning low or high PPO activity. ANOVA showed significant genotypic effects on PPO activity (P < 0.0001) in all populations. The generations and generation × genotype effects were not significant in any population. A putative third (null) genotype at Ppo-A1 (no PCR fragments for PPO18) was discovered in NW07OR1066 and NW07OR1070 derived populations, and these had the lowest mean PPO activities. Results demonstrated that both Ppo-A1 and Ppo-D1 loci affect the kernel PPO activity, but the Ppo-A1 has the major effect. In three populations, contrary results were observed to those predicted from previous work with Ppo-D1 alleles, suggesting the markers for Ppo-D1 allele might give erroneous results in some genetic backgrounds or lineages. Results suggest that selection for low or null alleles only at Ppo-A1 might allow development of low PPO wheat cultivars.

Effects of Single and Double Infections of Winter Wheat by Triticum mosaic virus and Wheat streak mosaic virus on Yield Determinants.

Triticum mosaic virus (TriMV) is a recently discovered virus infecting wheat (Triticum aestivum) in the Great Plains region of the United States. It is transmitted by wheat curl mites (Aceria tosichella) which also transmit Wheat streak mosaic virus (WSMV) and Wheat mosaic virus. In a greenhouse study, winter wheat 'Millennium' (WSMV susceptible) and 'Mace' (WSMV resistant) were mechanically inoculated with TriMV, WSMV, TriMV+WSMV, or sterile water at the two-leaf growth stage. At 28 days after inoculation, final chlorophyll meter (soil plant analysis development [SPAD]) readings, area under the SPAD progress curve (AUSPC), the number of tillers per plant, shoot and root weight, and total nitrogen and carbon content were determined. In Millennium, all measured variables were significantly reduced by single or double virus infections, with the greatest reductions occurring in the double-infection treatment. In Mace, only final SPAD readings, AUSPC, and total nitrogen were significantly reduced by single or double virus infections. There was a significant (P ≤ 0.05), positive linear relationship between SPAD readings and shoot weight in Millennium but not in Mace. The relationship between total nitrogen and shoot weight was positive, linear, and significant in both cultivars. The results from this study indicate that Mace, a WSMV-resistant cultivar, is also resistant to TriMV, and double infection of winter wheat by TriMV and WSMV exacerbates symptom expression and loss of biomass in susceptible cultivars.

The use of microsatellite markers for the detection of genetic similarity among winter bread wheat lines for chromosome 3A.

Previous studies with chromosome substitution and recombinant inbred chromosome lines identified that chromosome 3A of wheat cv. Wichita contains alleles that influence grain yield, yield components and agronomic performance traits relative to alleles on chromosome 3A of Cheyenne, a cultivar believed to be the founder parent of many Nebraska developed cultivars. This study was carried out to examine the genetic similarity among wheat cultivars based on the variation in chromosome 3A. Forty-eight cultivars, two promising lines and four substitution lines (in duplicate) were included in the study. Thirty-six chromosome 3A-specific and 12 group-3 barley simple sequence repeat (SSR) primer pairs were used. A total of 106 polymorphic bands were scored. Transferability of barley microsatellite markers to wheat was 73%. The coefficient of genetic distance (D) among the genotypes ranged from 0.40 to 0.91 and averaged D=0.66. Cluster analysis by the unweighted pair-group method with arithmetic averages showed one large and one small cluster with eight minor clusters in the large cluster. Several known pedigree relationships largely corresponded with the results of SSR clusters and principal coordinate analysis. Cluster analysis was also carried out by using 22 alleles that separate Wichita 3A from Cheyenne 3A, and three clusters were identified (a small cluster related to Cheyenne of mainly western Nebraska wheat cultivars; a larger, intermediate cluster with many modern Nebraska wheat cultivars; a large cluster related to Wichita with many modern high-yielding or Kansas wheat cultivars). Using three SSR markers that identify known agronomically important quantitative trait loci (QTL) regions, we again separated the cultivars into three main clusters that were related to Cheyenne or Wichita, or had a different 3A lineage. These results suggest that SSR markers linked to agronomically important QTLs are a valuable asset for estimating both genetic similarity for chromosome 3A and how the chromosome has been used in cultivar improvement.

Demarcating the gene-rich regions of the wheat genome.

By physically mapping 3025 loci including 252 phenotypically characterized genes and 17 quantitative trait loci (QTLs) relative to 334 deletion breakpoints, we localized the gene-containing fraction to 29% of the wheat genome present as 18 major and 30 minor gene-rich regions (GRRs). The GRRs varied both in gene number and density. The five largest GRRs physically spanning <3% of the genome contained 26% of the wheat genes. Approximate size of the GRRs ranged from 3 to 71 Mb. Recombination mainly occurred in the GRRs. Various GRRs varied as much as 128-fold for gene density and 140-fold for recombination rates. Except for a general suppression in 25-40% of the chromosomal region around centromeres, no correlation of recombination was observed with the gene density, the size, or chromosomal location of GRRs. More than 30% of the wheat genes are in recombination-poor regions thus are inaccessible to map-based cloning.

Transferability of SSR markers among wheat, rye, and triticale.

Simple sequence repeat (SSR) markers are a valuable tool for many purposes, such as mapping, fingerprinting, and breeding. However, they are only available in some economically important crops because of the high cost and labor intensity involved in their development. Comparative mapping reveals a high degree of colinearity between closely related species, which allows the exchange of markers between them. Our objective was to examine the transferability of SSR markers among wheat ( Triticum aestivum L.), rye ( Secale cereale L.), and triticale (X Triticosecale Wittmack). One hundred forty-eight wheat and 28 rye SSR markers were used to amplify genomic DNA extracted from five lines each of wheat, rye, and triticale. Transferability of wheat SSR markers to rye was 17%, whereas 25% of rye markers were amplifiable in wheat. In triticale, 58% and 39% transferability was achieved for wheat and rye markers, respectively. Wheat markers gave an average of 2.6, 2.7, and 2.4 polymorphic bands in wheat, rye, and triticale, respectively, while rye markers gave an average of 2.0 in rye and none in wheat and triticale. These transferable markers can now be exploited for further genetic and breeding studies in these species.

Virulence of Puccinia triticina on Wheat in Nebraska during 1997 and 1998.

Urediniospore isolates of Puccinia triticina were obtained from wheat leaf collections made in three wheat-growing regions in Nebraska in 1997 and in four regions in 1998. Using 16 Thatcher lines that are near-isogenic for leaf rust resistance, 17 virulence phenotypes were found among 121 single uredinial isolates in 1997, and 42 virulence phenotypes were found among 178 isolates in 1998. The most prevalent phenotype in 1997 was MDRR (virulent on Lr1, 3, 3ka, 10, 11, 18, 23, 24, and 30). In 1998, virulence phenotypes MDRR and MDRM (virulent on Lr1, 3, 3ka, 10, 11, 23, 24, and 30) were the most prevalent. In both years, virulence frequency was above 80% to genes Lr1, 3, 3ka, 10, 11, 23, 24, and 30 and below 21% to Lr2a, 17, and 26. Virulence frequency to Lr2c was 37% in 1997 and 22% in 1998. No virulence was found to Lr9, 16, or 21 in either year. New virulence phenotypes were detected in 1998 that were not found in 1997. In 1998, virulence was less frequent on Lr2a, 2c, 3ka, 18, 24, and 26 and more frequent on Lr17 than in 1997.

Physiologic Specialization of Puccinia recondita f. sp. tritici in Nebraska During 1995 and 1996.

Field samples of Puccinia recondita f. sp. tritici, collected from four wheat-growing regions in Nebraska in 1995 and from three in 1996, were characterized for virulence. Twenty virulence phenotypes were identified in 1995 and 18 in 1996. Virulence phenotypes MBR-10,18 (virulent on Lr genes, 1, 3, 3ka, 10, 11, 18, and 30) and MDR-10,18 (virulent on Lr genes 1, 3, 3ka, 10, 11, 18, 24, and 30) were the most prevalent, with each phenotype comprising 21.6% of the isolates characterized in 1995. Of the 1995 isolates, 24% were virulent on 10 or more host genes. No virulence to Lr16 and Lr17 was detected. In 1996, virulence phenotype MBR-10,18 was the most prevalent and comprised 20.5% of the isolates characterized. Of the 1996 isolates, 33% were virulent on 10 or more host genes. All isolates in both years were virulent on Lr1, Lr3, and Lr10. New virulence phenotypes were detected in 1996 that were not detected in 1995. In 1996, virulence was more frequent on Lr2a, Lr16, and Lr17 and less frequent on Lr3ka, Lr18, Lr24, Lr26, and Lr30. The number of isolates virulent on Lr24 and Lr26 has decreased from 83 and 53%, respectively, in 1992, to 34 and 1%, respectively, in 1996.

Root tip cell cycle synchronization and metaphase-chromosome isolation suitable for flow sorting in common wheat (Triticum aestivum L.).

An efficient procedure for cell-cycle synchronization in meristematic root tips was achieved in common wheat. Treatment parameters for synchronizing the cell cycle of root tip meristem cells, such as time-course and applied concentrations of various chemicals, were systematically tested and optimized by flow cytometric analysis of isolated nuclei. High mitotic indices (69.5% in the root tip meristematic area) were routinely obtained by treating germinating seeds with 1.25 mM hydroxyurea for 16 h, followed by incubation in a hydroxyurea-free solution for 2 h, and treatment with 1 μM trifluralin for 4 h. Uniform seed germination prior to treatment is very important for achieving consistently high metaphase indices in the root tips. Large numbers of metaphase chromosomes, suitable for flow cytometric analysis and sorting, were isolated from synchronized root tip cells. Flow sorted wheat chromosomes, via univariate and bivariate analysis, showed four major chromosome peaks. Each discrete peak may represent wheat chromosome types with similar DNA content. Bivariate flow karyotyping based on AT and GC content did not improve the separation of wheat chromosomes.

Chromosomal locations of genes that control major RNA-degrading activities in common wheat (Triticum aestivum L.).

Seventeen RNA-degrading enzymes of common wheat, with apparent molecular masses from 42.2 kDa to 16.3 kDa, were observed by the RNA-SDS-PAGE assay. To determine their chromosome locations, all chromosome arms of common wheat except 4BS were assayed in their null condition by using a set of ditelosomic or nullitetrasomic lines of the cultivar Chinese Spring. Our results showed that only one chromosome location each was identified for the 22.8-kDa and the 21.2-kDa enzymes, as well as for the 21.6 kDa enzyme, and they are on chromosome arms 2AS and 2DS, respectively. Loci controlling the 20.1 kDa activity were on chromosome arms 2AL, 4BS, 4DS and 6BS. The locus or loci coding for the gene(s) of the 42.2-kDa, 40.9-kDa and 39.2-kDa enzymes were probably ocated on chromosome arm 5AS, and their expression, in agreement with most other RNA-degrading enzyme activities were stimulated when chromosome arm 5AL was missing, indicating a inhibiting locus on 5AL. Our data suggested that the 31.9-kDa, 30.6-kDa and 29.6-kDa enzymes were possibly products of a common precursor which might be coded by a gene(s) on chromosome arm 6BS, and that the processing is co-regulated by loci on chromosome arms 2BS, 3DS, 6AL, 6BL and 7BS. The remaining of the enzyme activities were consistently found in all of the lines tested, and thus are presumably encoded by multiple loci. The only other possibility is that, their loci may be on chromosome arm 4BS which we have not assayed in its null condition.

Wheat chromosome 2D carries genes controlling the activity of two DNA-degrading enzymes.

DNA-degrading enzymes of 24.0 kDa and 27.0 kDa were observed to have different activities in two common wheat (Triticum aestivum L.) cultivars, 'Wichita' and 'Cheyenne'. A substrate-based SDS-PAGE assay revealed that these two enzymes were much more active in 'Wichita' than in 'Cheyenne'. Genes controlling the activities of these two enzymes were localized on chromosome 2D by testing DNA-degrading activities in reciprocal chromosome substitution lines between 'Wichita' and 'Cheyenne'. While the allele on 'Wichita' chromosome 2D stimulated the activities of the 24.0- and 27.0-kDa enzymes in Cheyenne, the allele on 'Cheyenne' chromosome 2D did not reduce the activities of the 24-kDa and 27-kDa enzymes in 'Wichita'. Whether these genes code for the DNA-degrading enzymes themselves or for factors that regulate the enzyme activities remains unknown.

Identification, characterization, and comparison of RNA-degrading enzymes of wheat and barley.

RNA-degrading enzymes play an important role in regulating gene expression, and sequence analyses have revealed significant homology among several plant RNA-degrading enzymes. In this study we surveyed crude extracts of the above-ground part of the common wheat (Triticum aestivum L.) and the cultivated barley (Hordeum vulgare L.) for major RNA-degrading enzymes using a substrate-based SDS-PAGE assay. Fifteen wheat and fourteen barley RNA-degrading enzymes, with apparent molecular masses ranging from 16.3 to 40.1 kD, were identified. These RNA-degrading enzymes were characterized by their response to pH changes and addition of EDTA and ZnCl2 to the preincubation or incubation buffers. The 33.2- to 40.1-kD wheat and barley, 31.7-kD wheat, and 32.0-kD barley enzyme activities were inhibited by both zinc and EDTA and were relatively tolerant to alkaline environment. The 22.7- to 28.2-kD enzymes were inhibited by zinc but stimulated by EDTA. The 18.8-kD enzyme exists in both wheat and barley. It was active in an acid environment, was inhibited by zinc, but was not affected by EDTA. Two enzyme activities (31.0 and 32.0 kD) are unique to the common wheat.

The effects of interactions of culture environment with genotype on wheat (Triticum aestivum) anther culture response.

The interactions of genotype and several variables related to culture environment, including temperature pretreatment, conditioned medium and agar concentrations were examined in a series of experiments for their effects on percent anthers producing callus and number of embryoids produced per 100 anthers scored. Significant genotypic interaction was observed for both traits with all environmental variables except methods of medium conditioning. Such interactions involve both changes of response magnitude and changes of rank order of genotypes. The highest response frequencies observed were in excess of 30% of anthers callusing. Most lines examined responded relatively well to a culture regime utilizing a 4°C treatment for 7-14 d prior to anther excision, followed by float culture, without transfer and without preconditioning of the culture medium. The results indicate, however, that particular genotypes may have specific requirements with respect to various environmental conditions so that culture conditions may need to be adjusted, especially for the least responsive genotypes.

Production, morphology, and cytogenetic analysis of Elymus caninus (Agropyron caninum) x Triticum aestivum F1 hybrids and backcross-1 derivatives.

Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD) and wheatgrass, Etymus caninus (Agropyron caninum) (2n=4x=28, SSHH) - the first successful report of this cross. Reciprocal crosses and genotypes differed for percent seed set, seed development and F1 hybrid plant production. With E. caninus as the pollen parent, there was no hybrid seed set. In the reciprocal cross, seed set was 23.1-25.4% depending upon wheat genotype used. Hybrid plants were produced only by rescuing embryos 12-13 days post pollination with cv 'Chinese Spring' as the wheat parent. Kinetin in the medium facilitated embryo germination but inhibited root development and seedling growth. The hybrids were vigorous, self sterile, and intermediate between parents. These had expected chromosome number (2n=5x=35, ABDSH), very little chromosome pairing (0.51 II, 0.04 III) and some secondary associations. The hybrids were successfully backcrossed with wheat. Chromosome number in the BC1 derivatives varied 54-58 with 56 as the modal class. The BC1 derivatives showed unusually high number of rod bivalents or reduced pairing of wheat homologues. These were sterile and BC2 seed was produced using wheat pollen.