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Wheatgrass Thinopyrum intermedium is a source of agronomically valuable traits for common wheat. Partial wheat-wheatgrass amphidiploids and lines with wheatgrass chromosome substitutions are extensively used as intermediates in breeding programs. Line Agis 1 (6Agi2/6D) is present in the cultivar Tulaykovskaya 10 pedigree. Wheatgrass chromosome 6Agi2 carries multiple resistance to fungal diseases in various ecogeographical zones. In this work, we studied the transfer of chromosome 6Agi2 in hybrid populations Saratovskaya 29 × skaya 10 (S29 × T10) and Tulaykovskaya 10 × Saratovskaya 29 (T10 × S29). Chromosome 6Agi2 was identif ied by PCR with chromosome-specif ic primers and by genomic in situ hybridization (GISH). According to molecular data, 6Agi2 was transmitted to nearly half of the plants tested in the F2 and F3 generations. A new breeding line 49-14 (2n = 42) with chromosome pair 6Agi2 was isolated and characterized in T10 × S29 F5 by GISH. According to the results of our f ield experiment in 2020, the line had high productivity traits. The grain weights per plant (10.04 ± 0.93 g) and the number of grains per plant (259.36 ± 22.49) did not differ signif icantly from the parent varieties. The number of grains per spikelet in the main spike was signif icantly higher than in S29 ( p ≤ 0.001) or T10 ( p ≤ 0.05). Plants were characterized by the ability to set 3.77 ± 0.1 grains per spikelet, and this trait varied among individuals from 2.93 to 4.62. The grain protein content was 17.91 %, and the gluten content, 40.55 %. According to the screening for fungal disease resistance carried out in the f ield in 2018 and 2020, chromosome 6Agi2 makes plants retain immunity to the West Siberian population of brown rust and to dominant races of stem rust. It also provides medium resistant and medium susceptible types of response to yellow rust. The possibility of using lines/varieties of bread wheat with wheatgrass chromosomes 6Agi2 in breeding in order to increase protein content in the grain, to confer resistance to leaf diseases on plants and to create multif lowered forms is discussed.Пырей промежуточный Thinopyrum intermedium является источником агрономически ценных признаков для мягкой пшеницы, для передачи которых используют частичные пшенично-пырейные амфидиплоиды и линии с замещением хромосомами пырея. С использованием линии Агис 1 создан сорт яровой мягкой пшеницы Тулайковская 5, который входит в родословную сорта Тулайковская 10. В геноме сорта хромосома пшеницы 6D замещена хромосомой пырея 6Agi2, несущей комплексную устойчивость к грибным заболеваниям в различных эколого-географических зонах. В данной работе изучен характер передачи хромосомы пырея 6Agi2 в гибридных популяциях сортов Саратовская 29 × Тулайковская 10 (С29 × Т10) и Тулайковская 10 × Саратовская 29 (Т10 × С29). Хромосома пырея 6Agi2 идентифицирована с помощью хромосомоспецифичных праймеров и методом геномной in situ гибридизации. Согласно молекулярному анализу, хромосома 6Agi2 передавалась почти половине изученных растений в F2 и F3 поколениях. В F5 поколении Т10 × С29 с помощью GISH выделена и охарактеризована новая селекционная линия 49-14 (2n = 42) с парой хромосом 6Agi2. По результатам эксперимента в полевых условиях 2020 г. линия имела высокие показатели продуктивности. Масса зерен с растения (10.04 ± 0.93 г) и число зерен с растения (259.36 ± 22.49) достоверно не отличались от родительских сортов. Число зерен на колосок в главном колосе у линии 49-14 было достоверно выше, чем у сортов С29 (при р ≤ 0.001) и Т10 (при р ≤ 0.05). Растения характеризовались способностью завязывать 3.77 ± 0.1 зерна на колосок, размах изменчивости признака варьировал от 2.93 до 4.62 у индивидуальных растений. Содержание белка в зерне составило 17.91 %, клейковины – 40.55 %. Согласно скринингу на устойчивость к грибным болезням, проведенному в полевых условиях 2018 и 2020 гг., хромосома 6Agi2 сохраняет у растений иммунность к западносибирской популяции бурой ржавчины и к доминантным расам стеблевой ржавчины, а также обеспечивает средний устойчивый и средний восприимчивый типы реакции к возбудителям желтой ржавчины. Обсуждается возможность использования линий/сортов мягкой пшеницы, несущих хромосомы пырея 6Agi2, в селекции на увеличение содержания белка в зерне, на устойчивость к листостебельным заболеваниям и на создание многоцветковых форм.

Thinopyrum intermedium (2n = 6x = 42, JJJSJSStSt) has been hybridized extensively with common wheat and proven to be a valuable germplasm source for improving disease resistance and yield potential of wheat. A novel disease-resistant wheat-Th. intermedium double substitution line X479, carrying 1St(1B) and 4St-4JS (4B), was identified using multi-color non-denaturing fluorescence in situ hybridization (ND-FISH). With the aim of transferring Thinopyrum-specific chromatin to wheat, a total of 573 plants from F2 and F3 progenies of X479 crossed with wheat cultivar MY11 were developed and characterized using sequential ND-FISH with multiple probes. Fifteen types of wheat-Thinopyrum translocation chromosomes were preferentially transmitted in the progenies, and the homozygous wheat-1St, and wheat-4JSL translocation lines were identified using ND-FISH, Oligo-FISH painting and CENH3 immunostaining. The wheat-4JSL translocation lines exhibited high levels of resistance to stripe rust prevalent races in field screening. The gene for stripe rust resistance was found to be physically located on FL0–0.60 of the 4JSL, using deletion lines and specific DNA markers. The new wheat-Th. intermedium translocation lines can be exploited as useful germplasms for wheat improvement.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. Xiaoyan 78829, a partial amphidiploid developed by crossing common wheat with Thinopyrum intermedium, is immune to wheat stripe rust. To transfer the resistance gene of this excellent germplasm resource to wheat, the translocation line WTT11 was produced by pollen irradiation and assessed for immunity to stripe rust races CYR32, CYR33 and CYR34. A novel stripe rust-resistance locus derived from Th. intermedium was confirmed by linkage and diagnostic marker analyses. Molecular cytogenetic analyses revealed that WTT11 carries a TTh·2DL translocation. The breakpoint of 1B was located at 95.5 MB, and the alien segments were found to be homoeologous to wheat-group chromosomes 6 and 7 according to a wheat660K single-nucleotide polymorphism (SNP) array analysis. Ten previously developed PCR-based markers were confirmed to rapidly trace the alien segments of WTT11, and 20 kompetitive allele-specific PCR (KASP) markers were developed to enable genotyping of Th. intermedium and common wheat. Evaluation of agronomic traits in two consecutive crop seasons uncovered some favorable agronomic traits in WTT11, such as lower plant height and longer main panicles, that may be applicable to wheat improvement. As a novel genetic resource, the new resistance locus may be useful for wheat disease-resistance breeding.

Wide hybridization between wheat and wild relatives such as Thinopyrum intermedium is important for broadening genetic diversity and improving disease resistance in wheat. We developed 30 wheat-Th. intermedium derivatives. Here, we report assessments of their resistance to different pathogens including cereal cyst nematode (CCN; Heterodera spp.), Puccinia striiformis f. tritici Erikss. causing stripe rust, and Blumeria graminis f. tritici (DC.) Speer inciting powdery mildew. Under natural field infection, all the wheat-Th. intermedium lines were resistant to at least one of the pathogens, and four lines were resistant to multiple pathogens. Twenty-nine of 30 tested lines exhibited resistance to H. avenae, a dominant CCN species in wheat fields. Twenty-four lines were resistant to H. filipjevi, an emerging threat to wheat production. Tests of phenotypic responses in the naturally infected field nurseries identified six stripe rust-resistant lines and 13 powdery mildew-resistant lines. Mitotic observation demonstrated that these newly developed wheat-Th. intermedium derivatives included not only octoploid but also chromosome addition, substitution, and translocation lines. Chromosome compositions of the four lines resistant to multiple pathogens were analyzed by genomic in situ hybridization and fluorescence in situ hybridization. The octoploid lines Zhong 10-68 and Zhong 10-117 carried both intact Th. intermedium chromosomes and translocated chromosomes. Line Zhong 10-149 had 42 wheat chromosomes and two wheat ditelosomes plus a pair of T3BS·J translocated chromosomes. Line Zhong 10-160 carried 41 wheat chromosomes plus one pair of the J genome chromosomes of Th. intermedium. The multiple disease-resistant wheat-Th. intermedium derivatives, especially lines with chromosome counts close to that of common wheat, provide valuable materials for wheat resistance breeding programs.

Thinopyrum intermedium has been used as a resource for improving resistance to biotic and abiotic stresses and yield potential in common wheat. Wheat line SN304 was derived from a cross between common wheat cultivar Yannong 15 and Th. intermedium. Genomic in situ hybridization (GISH) produced no hybridization signal in SN304 using Th. intermedium genomic DNA as a probe, but fluorescence in situ hybridization (FISH) using oligonucleotides AFA-3, AFA-4, pAs1-1, pAs1-3, pAs1-4, pAs1-6, pSc119.2-1, and (GAA)10 as probes detected hybridization signals on chromosomes 2A, 7A, 2B, 3B, 6B, and 7B in SN304 that differed from Yannong 15. Results of specific markers also indicated that there were Th. intermedium chromatin introgressions on different chromosomes in SN304. In a hydroponic culture experiment, SN304 not only produced more biomass and higher stem and leaf dry weight but also accumulated more phosphorus than Yannong 15 under phosphorus-deficiency stress. Moreover, SN304 produced a lower pH and released more organic acids, especially oxalic acid, than Yannong 15, which suggests that SN304 exudates enabled more absorbance of P than Yannong 15 under comparable conditions. The results indicate that SN304 is a wheat-Th. intermedium introgression line with tolerance to phosphorus-deficiency stress.

Thinopyrum intermedium possesses many desirable agronomic traits that make it a valuable genetic source for wheat improvement. The precise identification of individual chromosomes of allohexaploid Th. intermedium is a challenge due to its three sub-genomic constitutions with complex evolutionary ancestries. The non-denaturing fluorescent in situ hybridization (ND-FISH) using tandem-repeat oligos, including Oligo-B11 and Oligo-pDb12H, effectively distinguished the St, J and JS genomes, while Oligo-FISH painting, based on seven oligonucleotide pools derived from collinear regions between barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.), was able to identify each linkage group of the Th. intermedium chromosomes. We subsequently established the first karyotype of Th. intermedium with individual chromosome recognition using sequential ND-FISH and Oligo-FISH painting. The chromosome constitutions of 14 wheat–Th. intermedium partial amphiploids and addition lines were characterized. Distinct intergenomic chromosome rearrangements were revealed among Th. intermedium chromosomes in these amphiploids and addition lines. The precisely defined karyotypes of these wheat–Th. intermedium derived lines may be helpful for further study on chromosome evolution, chromatin introgression and wheat breeding programs.

Spring wheat is one of the widely cultivated crops in the Russian Federation. Wheat breeding is aimed at creation of varieties characterized by high productivity and grain quality. When creating new varieties, the main attention is paid to ecological stability to environments, which negatively affects the agromomic traits and yield. Currently there is no enough information on the inheritance and manifestation of productivity traits in various agro-ecological conditions in advanced generations of the crosses between winter and spring wheat varieties. In this study, we analyzed the variability of the yield traits in wheat breeding lines (Triticum aestivum L.) of F6-7 generations, obtained from hybridization of winter wheat varieties with spring donors of leaf rust resistance genes with the aim to identify promising genotypes as ф source of valuable agronomic traits. Field trials were performed in 2018 in three regions, the Novosibirsk and Omsk region and the Republic of Tatarstan. The following traits were studied: tiller number per plant, the grain number per ear, the grain weight per ear, 1000 grain weight, and the grain weight per plant. Ecological plasticity of the lines was studied using indexes of the intensity and stability. Analysis of variance based on the results of field evaluation indicates a significant influence of the genotype, the environment and their interaction in the phenotypic manifestation of all the studied characters. The highest contribution of the genotype is shown for the grain number per ear (42.8 %) and the 1000 grain weight (57.0 %). A high contribution of environmental factors was found for tiller number (41.8 %) and grain weight per ear (40.3 %). The genotype ½ environment interaction had a significant effect on all traits, its contribution varied from 25.9 % (1000 grain weight) to 41.0 % (grain weight per ear), which indicates a significant response of genotypes to changing climatic factors. The results of field tests showed that there was a high variability of all characters in all three climatic zones, but the degree of variation differed. The averaged indicators of the studied traits were lower in the field conditions of the Omsk zone as compared to the Novosibirsk region and Tatarstan. Higher fluctuations were noted for the grain number per ear (13.0-69.0), the grain weight per ear (0.35-2.65 g), and the grain weight per plant (0.15-6.95) in Tatarstan’s environments in comparison with other regions. The estimation of stability and intensity indices showed that 16 of 55 genotypes have intensive type, 35 were semi-intensive and 4 were extensive. The grouping of samples by the principal coordinate analysis method divided the genotypes into four main clusters according to the stability and intensity parameters. Molecular analysis for the presence of leaf rust resistance genes Lr6Ai#2, LrAsp5, and LrTt2 introduced from Thinopyrum intermedium (Host) Barkworth & D.R. Dewey, Aegilops speltoides Tausch and Triticum timopheevii Zhuk., respectively, showed that 10 out of 55 lines do not contain alien genetic material. The results of marker analysis for the presence of resistance genes did not correlate with the clustering of samples by intensity and stability types. This fact suggests that the presence of alien genomes does not influence on the stability of breeding lines. Based on the obtained results, genotypes with valuable characters were selected as sources of productivity.

Многолетняя пшеница — трититригия, несущая гены своих родительских видов, в качестве которых использовали виды пырея Agropyron elongatum (Host) P. Beauv. [syn. Thinopyrum ponticum (Podp.) Zhi W. Liu et R. R.-C. Wang] и A. glaucum (Desf. ex DC) Roem. and Schult. [syn. Thinopyrum intermedium (Host) Barkworth et D. R. Dewey] и сорта мягкой и твердой пшеницы (Triticum aestivum L. и T. durum Desf.) в различных комбинациях, унаследовала много положительных свойств своих родителей (многолетность, способность к отрастанию после уборки, более высокий показатели качества зерна — содержание белка, клейковины и т.д., устойчивость к неблагоприятным почвенно-климатическим условиям, ряду болезней). Для установления объективной оценки качественных показателей зерна трититригии в условиях центрального региона России в зоне рискованного земледелия изучены технологические и хлебопекарные показатели некоторых образцов данной культуры. При обычных условиях для роста и развития растений многолетняя пшеница накапливает в зерне значительное количество белка — 18 – 19 %. Анализ хлебопекарных достоинств трититригии показал, что основные показатели такие как внешний вид, пористость, объем хлеба, превышают показатели сорта озимой пшеницы Московская 39, используемого в качестве стандарта. Исследуемые образцы многолетней пшеницы (М 3202, ЗП 26,548, Памяти Любимовой) имели высокую хлебопекарную оценку — от 3,8 до 4,0 баллов соответственно, тогда как у стандарта Московская 39 общая оценка — 3,7 балла.

Premise Nucleic acid integrity can be compromised under many abiotic stresses. To date, however, few studies have considered whether nucleic acid damage and damage repair play a role in cold-stress adaptation. A further insufficiently explored question concerns how age affects cold stress adaptation among mature perennials. As a plant ages, the optimal trade-off between growth and stress tolerance may shift. Methods Oxidative damage to RNA and expression of genes involved in DNA repair were compared in multiple mature cohorts of Thinopyrum intermedium (an emerging perennial cereal) and in wheat and barley under intermittent freezing stress and under nonfreezing conditions. Activity of glutathione peroxidase (GPX) and four other antioxidative enzymes was also measured under these conditions. DNA repair genes included photolyases involved in repairing ultraviolet-induced damage and two genes involved in repairing oxidatively induced damage (ERCC1, RAD23). Results Freezing stress was accompanied by large increases in photolyase expression and ERCC1 expression (in wheat and Thinopyrum) and in GPX and GR activity (particularly in Thinopyrum). This is the first report of DNA photolyases being overexpressed under freezing stress. Older Thinopyrum had lower photolyase expression and less freezing-induced overexpression of ERCC1. Younger Thinopyrum plants sustained more oxidative damage to RNA. Conclusions Overexpression of DNA repair genes is an important aspect of cold acclimation. When comparing adult cohorts, aging was associated with changes in the freezing stress response, but not with overall increases or decreases in stress tolerance.

Kernza® intermediate wheatgrass [Thinopyrum intermedium (Host) Barkworth & Dewey], the first perennial grain crop to come to market in North America, can provide a number of ecosystem services when integrated into cropping systems that are dominated by annual grain crops. However, grain yield from Kernza is lower than comparable annual cereal crops such as wheat and oats. Also, although Kernza is a long-lived perennial that can persist for decades, grain yield tends to decline over time as Kernza stands age leading most farmers to replant or rotate to a different crop after 3–5 yrs. Increased intraspecific competition as stand density increases with age has been reported to cause grain yield declines. We investigated the effect of strip-tillage applied at two different timings, between the third and fourth grain harvests, from a Kernza stand in upstate New York. Strip-tillage applied in late fall as plants were entering dormancy increased grain yield by 61% when compared to the control treatment without strip-tillage. However, total crop biomass was not reduced resulting in a greater harvest index for the fall strip-tillage treatment. Strip-tillage applied before stem elongation the following spring reduced overall tiller density and total crop biomass but did not impact tiller fertility or grain yield compared to the control treatment without strip-tillage. Increased grain yield in the fall strip-tillage treatment was due to an increase in the percentage of tillers that produced mature seedheads. This suggests that grain yield decline over time is at least partially caused by competition between tillers in dense stands. Results support further research and development of strip-tillage and other forms of managed disturbance as tools for maintaining Kernza grain yield over time.

Thinopyrum intermedium (2n = 6x = 42, E1E1E2E2XX) serves as an important gene source of desirable traits for genetic improvement of wheat cultivars resistant to stresses. This study used the comparative proteomic approach to identify stress defense related proteins in the developing grains of common wheat (Zhongmai 8601)-Thinopyron intermedium 7XL/7DS translocation line YW642 and to explore their potential values for improving wheat stress resistance. Two-dimensional electrophoresis identified 124 differentially accumulated protein spots representing 100 unique proteins, which mainly participated in stress defense, energy metabolism, protein metabolism and folding and storage protein synthesis. Among these, 16 were unique and 35 were upregulated in YW642. The upregulated DAPs were mainly involved in biotic and abiotic stress defense. Further cis-elements analysis of these stress-related DAP genes revealed that phytohormone responsive elements such as ABREs, G-box, CGTCA-motif and TGACG-motif, and environment responsive element As-1 were particularly abundant, which could play important roles in response to various stressors. Transcription expression analysis by RNA-seq and qRT-PCR demonstrated a large part of the stress-related DAP genes showed an upregulated expression in the early-to-middle stages of grain development. Our results proved that Thinopyron intermedium contains abundant stress responsive proteins that have potential values for the genetic improvement of wheat stress resistance.

Estimation of spelt wheat and the species of intermediate wheatgrass by the main agrobiological indicators (calendar dates of the beginning of the main phases of plant development, plant height dynamics, dry mass accumulation, grain yield and protein content) compared to soft wheat was done. The index of stability of grain yield formation was determined. It was found that the main phases of development in spelt wheat occurred on average 10–15 days later than in soft wheat. Plants of intermediate wheatgrass in the first year of cultivation had slower growth. The main phases of development in plants of intermediate wheatgrass of the second and third year of growth occurred almost in the same time with soft wheat. It was established that spelt wheat and intermediate wheatgrass were significantly superior to soft wheat in plant height. In the earing phase, the height of these plants was on average more than 100 cm, which had to be taken into account when growing these crops. It varied from 25 to 42 cm in spelt wheat and from 29 to 57 cm in intermediate wheatgrass during the stem elongation phase depending on the weather conditions of the research year. This indicator ranged from 107 to 113 cm and from 96 to 117 cm, respectively, in the earing phase, and from 137 to 168 cm and from 107 to 196 cm in the phase of milk ripeness of grain. Plants of spelt wheat and intermediate wheatgrass had a high stability index of the formation of dry matter and grain yield. It should be noted that intermediate wheatgrass formed a significant vegetative mass even in the earing phase. The vegetative mass in spelt wheat was formed during the earing period – full ripeness of grain. These crops (spelt wheat – 22.1 ± 0.4, intermediate wheatgrass – 22.4–24.6 ± 0.2–0.5) were significantly superior to soft wheat (12.1 ± 0.5) by the content of protein in grain, so it was recommended to involve them in the selection programs to create species with high productivity. However, spelt wheat (5.58 ± 0.13) was less and intermediate wheatgrass (0.98–1.22 ± 0.14–0.18) was the most inferior to soft wheat (8.03 ± 0.27) in terms of grain yield. Keywords: Spelt wheat; intermediate wheatgrass; plant height; plant development phases; dry mass; yield; protein References Hendrickson, J. R. (2014). Tillage and grazing impact on annual crop yields following conversion from perennial grass to annual crops. Crop Management, 13(1), 613–627. Bajgain, P., Zhang, X., Jungers, J., DeHaan, L., Heim, B., Sheaffer, C., Wyse, D., Anderson, J. (2020). ‘MN???Clearwater’, the first food???grade intermediate wheatgrass (Kernza perennial grain) cultivar. Journal of Plant Registrations. Zhong, Y., Mogoginta, J., Gayin, J., Annor, G. (2019). Starch hydrolysis kinetics of intermediate wheatgrass (Thinopyrum intermedium) flour and its effects on the unit chain profile of its resistant starch fraction. Cereal Chemistry, 96(3). 564–574. doi: 10.1002/cche.10156. 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Key Message Paternity assignment and genome-wide association analyses for fertility were applied to aThinopyrum intermediumbreeding program. A lack of progeny between combinations of parents was associated with loci near self-incompatibility genes. Abstract In outcrossing species such as intermediate wheatgrass (IWG, Thinopyrum intermedium), polycrossing is often used to generate novel recombinants through each cycle of selection, but it cannot track pollen-parent pedigrees and it is unknown how self-incompatibility (SI) genes may limit the number of unique crosses obtained. This study investigated the potential of using next-generation sequencing to assign paternity and identify putative SI loci in IWG. Using a reference population of 380 individuals made from controlled crosses of 64 parents, paternity was assigned with 92% agreement using Cervus software. Using this approach, 80% of 4158 progeny (n = 3342) from a polycross of 89 parents were assigned paternity. Of the 89 pollen parents, 82 (92%) were represented with 1633 unique full-sib families representing 42% of all potential crosses. The number of progeny per successful pollen parent ranged from 1 to 123, with number of inflorescences per pollen parent significantly correlated to the number of progeny (r = 0.54, p

Stripe rust is an important disease in wheat, and development of genetic resistance in cultivars is an effective approach to control the disease. Wild species of wheat, such as Thinopyrum intermedium, are an excellent gene source for wheat improvement. In this study, two stripe rust-resistant wheat–Th. intermedium chromosome translocation lines, CH4131 and CH4132, were characterized by cytogenetic and pathological methods. The introgressed chromosome fragment was tagged using amplified fragment-length polymorphism-derived sequence-characterized amplified region (SCAR) markers and intron targeting markers, indicating that CH4131 and CH4132 both possess a homologous group 3 chromatin of Th. intermedium. Genomic in situ hybridization results suggested that a very small Th. intermedium chromosome segment was translocated to the terminal region of wheat 1BS for both lines, forming a configuration of T3Ai-1BS.1BL. The two translocation lines were resistant to stripe rust, and the resistance gene, temporarily designated YrCH-1BS, was likely derived from Th. intermedium. The translocated chromosome fragments have no genetic linkage drag to agronomic performance. The grain quality indexes of these two translocations were higher than local wheat varieties. Therefore, CH4131 and CH4132 could be used as potential gene sources in wheat improvement programs. The SCAR markers are useful to select stripe rust resistance from Th. intermedium.

The development of next generation sequencing (NGS) enabled a shift from array-based genotyping to high-throughput genotyping by directly sequencing genomic libraries. Even though whole genome sequencing was initially too costly for routine analysis in large populations, such as those utilized for breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to utilize whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage, a limitation comes in the time and high cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on amount of data required and extended to 3,072 samples or more. Panels of double haploid wheat lines (Triticum aestivum, CDC Stanley x CDC Landmark), wheat-barley (T. aestivum x Hordeum vulgare) and wheat-wheatgrass (Triticum durum x Thinopyrum intermedium) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1x down to 0.01x per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the low coverage skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

Estimation of spelt wheat and the species of intermediate wheatgrass by the main agrobiological indicators (calendar dates of the beginning of the main phases of plant development, plant height dynamics, dry mass accumulation, grain yield and protein content) compared to soft wheat was done. The index of stability of grain yield formation was determined. It was found that the main phases of development in spelt wheat occurred on average 10–15 days later than in soft wheat. Plants of intermediate wheatgrass in the first year of cultivation had slower growth. The main phases of development in plants of intermediate wheatgrass of the second and third year of growth occurred almost in the same time with soft wheat. It was established that spelt wheat and intermediate wheatgrass were significantly superior to soft wheat in plant height. In the earing phase, the height of these plants was on average more than 100 cm, which had to be taken into account when growing these crops. It varied from 25 to 42 cm in spelt wheat and from 29 to 57 cm in intermediate wheatgrass during the stem elongation phase depending on the weather conditions of the research year. This indicator ranged from 107 to 113 cm and from 96 to 117 cm, respectively, in the earing phase, and from 137 to 168 cm and from 107 to 196 cm in the phase of milk ripeness of grain. Plants of spelt wheat and intermediate wheatgrass had a high stability index of the formation of dry matter and grain yield. It should be noted that intermediate wheatgrass formed a significant vegetative mass even in the earing phase. The vegetative mass in spelt wheat was formed during the earing period – full ripeness of grain. These crops (spelt wheat – 22.1 ± 0.4, intermediate wheatgrass – 22.4–24.6 ± 0.2–0.5) were significantly superior to soft wheat (12.1 ± 0.5) by the content of protein in grain, so it was recommended to involve them in the selection programs to create species with high productivity. However, spelt wheat (5.58 ± 0.13) was less and intermediate wheatgrass (0.98–1.22 ± 0.14–0.18) was the most inferior to soft wheat (8.03 ± 0.27) in terms of grain yield. Keywords: Spelt wheat; intermediate wheatgrass; plant height; plant development phases; dry mass; yield; protein References Hendrickson, J. R. (2014). Tillage and grazing impact on annual crop yields following conversion from perennial grass to annual crops. Crop Management, 13(1), 613–627. Bajgain, P., Zhang, X., Jungers, J., DeHaan, L., Heim, B., Sheaffer, C., Wyse, D., Anderson, J. (2020). ‘MN�?�Clearwater’, the first food�?�grade intermediate wheatgrass (Kernza perennial grain) cultivar. Journal of Plant Registrations. Zhong, Y., Mogoginta, J., Gayin, J., Annor, G. (2019). Starch hydrolysis kinetics of intermediate wheatgrass (Thinopyrum intermedium) flour and its effects on the unit chain profile of its resistant starch fraction. Cereal Chemistry, 96(3). 564–574. doi: 10.1002/cche.10156. 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Thinopyrum intermedium (2n = 6x = 42, JJJsJsStSt) has been hybridized extensively with common wheat and has proven to be a valuable germplasm source for improving disease resistance, quality attributes, and yield potential in wheat. We characterized new disease resistant wheat-Th. intermedium derivatives A1082 and A5-5 using sequential multi-color fluorescence in situ hybridization (mc-FISH), genomic in situ hybridization (GISH), PCR-based landmark unique gene (PLUG) and intron targeting (IT) markers. A1082 was identified as a wheat-Th. intermedium 3J disomic addition line, and A5-5 was a T4BS·5JsL homozygous Robertsonian translocation line. Seventy-one and 106 pairs of primers amplified Th. intermedium-specific bands allowing chromosomes 3J and 5Js to be tracked, respectively. A new oligonucleotide probe, Oligo-6H-2-100, was developed for FISH labeling of the subterminal region of the long arm of chromosome 5Js. Both lines were highly resistant to stripe rust pathogen races prevalent in Chinese field screening nurseries. A5-5 also displayed a significant increase in tiller number compared to its wheat parent. The new lines can be exploited as useful germplasms for wheat improvement. Keywords: Disease resistance, FISH, GISH, Stripe rust, Triticum aestivum

The global interest in growing perennial grain crops such as intermediate wheatgrass (Thinopyrum intermedium) (Kernza) for production of food and feed is increasing. Intercropping Kernza with legum...

Powdery mildew caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt) is an econo-mically important disease of common wheat T. aestivum L. One of the most effective and environmentally important ways of protection of wheat against Bgt is cultivation of the varieties with genetic resistance. The aim of this work was to study the genetic diversity of Russian spring wheat varieties for the powdery mildew resistance loci. Phytopathological evaluation of 97 wheat varieties showed that no more than 10% of the varieties have low level of susceptibility to the Bgt population specific to the Western Siberian region. Association mapping carried out on the basis of SNP genotyping and phytopathological testing during three environmental seasons identified eight loci in chromosomes 1AL, 1DS, 2BL, 5AS, 5DS, 6AL, 6DL, and 7AL. A high impact to the phenotypic manifestation of the trait was established for genetic factors localized in chromosomes 5AS, 6AL, and 6DL. The long arm of chromosome 6D contains the gene Pm6Ai=2, which was introduced from wheatgrass Thinopyrum intermedium and provides effective protection against the powdery mildew pathogen. On the basis of comparative analysis of the chromosomal localization of the known Pm resistance genes and loci mapped in this work, it was assumed that QTLs in chromosomes 1DS, 5AS, and 6AL are novel, not previously described resistance loci. The obtained results can be used in breeding programs for selection of target loci and for development of molecular markers specific to Bgt resistance loci.

Barley yellow dwarf (BYD) is a major virus disease which dramatically reduces wheat yield. Introducing BYD resistance genes into commercial varieties has been proven to be effective in reducing damage caused by barley yellow dwarf virus (BYDV). However, only one major resistance gene is readily deployable for breeding; Bdv2 derived from Thinopyrum intermedium is deployed as a chromosomal translocation. In this study, a double haploid (DH) population was developed from a cross between XuBYDV (introduced from China showing very good resistance to BYD) and H-120 (a BYD-sensitive Chinese accession), and was used to identify QTL for BYD resistance. The population was genotyped using an Infinium iSelect bead chip array targeting 90K gene-based SNPs. The disease resistance of DH lines inoculated with BYDV was assessed at the heading stage. The infections were assessed by tissue blot immunoassay (TBIA). Three new QTL were identified on chromosomes 5A, 6A, and 7A for both symptom and TBIA, with all three resistance alleles being inherited from XuBYDV. Some DH lines with the resistance alleles from all three QTL showed high level resistance to BYD. These new QTL will be useful in breeding programs for pyramiding BYD resistance genes.

Aim. Thinopyrum intermedium , a wild wheat relative, for some reasons is on interest for its involvement to the introgressive hybridization with wheat. The basis for the purposeful work in this direction should be the information about the biological and genetic properties of the species, including the mating system. The mating system can be identified based on the genotyping of plants in populations for a polymorphic gene with codominant inheritance. Gene Glu was used as such a codominant gene. Methods. Cytological (determination of the chromosome number in metaphases of mitosis), electrophoresis of glutenin storage proteins, comparative analysis of electrophoretic spectra, and the population genetic methods for calculating the frequencies of genes and genotypes . Results. Plants were hexaploid, with 2n = 42 chromosomes. In a sample of fifty plants, a genetic control of components of the glutenin spectra has been established. They are controlled by three independent genes: Glu-Thi1-2 with four alleles 1-2 (cluster), 1, 2, 0, Glu-Thi1-4 withfour alleles 1-4 (cluster), 1, 4, 0, and the three-allele gene Glu-Thi1-3-5 . The participation of the three genes in the formation of the electrophoretic spectrum of glutenins is consistent with the hexaploid nature of Thinopyrum intermedium , which follows from the established number of 42 chromosomes. The information about the genetic control of glutenins is the basis for determining the mating system of this species. A simplified three-allele model of genetic control of glutenins’ electrophoretic spectra was used for determination of the mating system of Thinopyrum intermedium. Conclusions. Plants of the studied Thinopyrum intermedium population have a mixed mating system and the probability of self-pollination ranging from 0,50 to 0,81, depending on the Glu gene, which is used for determination of heterozygosity of the plants. This biological characteristic is positive for prospective use of this species for wide hybridization.

Intermediate wheatgrass (Thinopyrum intermedium, IWG) is a perennial grain crop with high biomass and grain yield, long seeds, and resistance to pests and diseases. It also reduces soil erosion, nitrate and mineral leaching into underground water tables, and sequesters carbon in its roots. The domestication timeline of IWG as a grain crop spans only 3 decades, hence it lags annual grain crops in yield and seed characteristics. One approach to improve its agronomic traits is by using molecular markers to uncover marker-trait associations. In this study, we performed association mapping on IWG breeding germplasm from the third recurrent selection cycle at the University of Minnesota. The IWG population was phenotyped in St Paul, MN in 2017 and 2018, and in Crookston, MN in 2018 for grain yield, seed length, width and weight, spike length and weight, and number of spikelets per spike. Strong positive correlations were observed among most trait pairs, with correlations as high as 0.76. Genotyping using high throughput sequencing identified 8,899 high-quality genome-wide SNPs which were combined with phenotypic data in association mapping to discover regions associated with the yield component traits. We detected 154 genetic loci associated with these traits of which 19 were shared between at least two traits. Prediction of breeding values using significant loci as fixed effects in genomic selection model improved predictive abilities by up to 14%. Genetic mapping of agronomic traits followed by using genomic selection to predict breeding values can assist breeders in selecting superior genotypes to accelerate IWG domestication.

Background Interspecific hybridisation resulting in polyploidy is one of the major driving forces in plant evolution. Here, we present data from the molecular cytogenetic analysis of three cytotypes of Elytrigia ×mucronata using sequential fluorescence (5S rDNA, 18S rDNA and pSc119.2 probes) and genomic in situ hybridisation (four genomic probes of diploid taxa, i.e., Aegilops, Dasypyrum, Hordeum and Pseudoroegneria). Results The concurrent presence of Hordeum (descended from E. repens) and Dasypyrum + Aegilops (descended from E. intermedia) chromosome sets in all cytotypes of E. ×mucronata confirmed the assumed hybrid origin of the analysed plants. The following different genomic constitutions were observed for E. ×mucronata. Hexaploid plants exhibited three chromosome sets from Pseudoroegneria and one chromosome set each from Aegilops, Hordeum and Dasypyrum. Heptaploid plants harboured the six chromosome sets of the hexaploid plants and an additional Pseudoroegneria chromosome set. Nonaploid cytotypes differed in their genomic constitutions, reflecting different origins through the fusion of reduced and unreduced gametes. The hybridisation patterns of repetitive sequences (5S rDNA, 18S rDNA, and pSc119.2) in E. ×mucronata varied between and within cytotypes. Chromosome alterations that were not identified in the parental species were found in both heptaploid and some nonaploid plants. Conclusions The results confirmed that both homoploid hybridisation and heteroploid hybridisation that lead to the coexistence of four different haplomes within single hybrid genomes occur in Elytrigia allopolyploids. The chromosomal alterations observed in both heptaploid and some nonaploid plants indicated that genome restructuring occurs during and/or after the hybrids arose. Moreover, a specific chromosomal translocation detected in one of the nonaploids indicated that it was not a primary hybrid. Therefore, at least some of the hybrids are fertile. Hybridisation in Triticeae allopolyploids clearly and significantly contributes to genomic diversity. Different combinations of parental haplomes coupled with chromosomal alterations may result in the establishment of unique lineages, thus providing raw material for selection. Electronic supplementary material The online version of this article (10.1186/s12870-019-1806-y) contains supplementary material, which is available to authorized users.

Thinopyrum intermedium and barley are two close relatives of wheat and carry many genes that are potentially valuable for the improvement of various wheat traits. In this study we created wheat double substitution lines by hybridizing different wheat–Th. intermedium and wheat–barley disomic alien substitution lines, with the aim of using genes in Th. intermedium and barley for wheat breeding and investigating the genetic behavior of alien chromosomes and their wheat homoeologs. As expected, we obtained two types of wheat double substitution lines, 2D2Ai#2(2B)2H(2A) and 2A2Ai#2(2B)2H(2D), in which different group 2 wheat chromosomes were replaced by barley chromosome 2H and Th. intermedium chromosome 2Ai#2. The new materials were characterized using molecular markers, genomic in situ hybridization (GISH), and fluorescent in situ hybridization (FISH). GISH and FISH experiments revealed that the double substitution lines harbor 42 chromosomes including 38 wheat chromosomes, a pair of barley chromosomes, and a pair of Th. intermedium chromosomes. Analysis using specific DNA markers showed that two pairs of wheat homoeologous group 2 chromosomes in the new lines were substituted by a pair of 2H and a pair of 2Ai#2 chromosomes. Chromosome 2H showed a higher transmission rate than 2Ai#2, and both chromosomes were preferentially transmitted between generations via female gametes. Evaluation of botanic and agronomic traits demonstrated that, compared with their parents, the new lines showed similar growth habits and plant type but differences in plant height, flowering date, and self-fertility. Cytological observations using different probes suggested that the double substitution lines showed nearly normal genetic behavior before and during meiosis. The novel substitution lines can potentially be used in wheat meiosis research and breeding programs. Keywords: Wheat, Double substitution lines, Molecular markers, Transmission rate of alien chromosomes, Genomic in situ hybridization

Cultivating perennial crops such as intermediate wheatgrass, (IWG) (Thinopyrum intermedium), benefits the environment, e.g. by reducing nitrogen run-off compared to cultivation of annual crops like wheat. However, IWG's ability to form a gluten-network is low, and processing modifications may be needed to improve it. Our objective was to study the effect of bran reduction and addition of dough conditioners (wheat protein isolate, vital wheat gluten, ascorbic acid, xylanase, transglutaminase) on extensibility, resistance to extension and protein secondary structures of IWG dough. Grain kernels were analyzed for dimensions and chemical composition. IWG from two locations was used, hard red wheat served as reference. IWG kernels were thinner, lighter, and of lower bulk density than wheat; flour contained more insoluble dietary fiber, protein and ash; and dough was less extensible. Growing location affected fiber and protein content, and was associated with structural and rheological differences. Bran addition increased β-sheets and resistance to extension, but decreased β-turns and extensibility. Transglutaminase increased resistance to extension and affected protein conformation, especially α-helices. Wheat proteins addition had similar, but less pronounced effects. Future studies to evaluate different levels and combinations of dough conditioners, and their effects on bread characteristics, are needed.

Emerging perennial grain crops yield less grain than annual crops, but the economic viability of these perennial systems could be improved if both forage and grain are harvested. However, the belowground consequences of forage removal in perennial grain systems are unknown. This study aimed to determine the effect of the additional harvest of forage biomass on overall plant biomass allocation and labile soil C and N dynamics within a perennial grain dual-use system. Plant biomass and associated soil samples of a perennial grain [Kernza (Thinopyrum intermedium)] were taken monthly over the first three growing seasons under three harvest regiments: No Cut (0x), Summer Cut (1x), and Summer and Fall Cut (2x). The harvesting of forage biomass significantly increased both above- and belowground biomass. The once and twice forage-harvested treatments averaged 39% and 73% greater root biomass in 2016 and 39% and 49% greater root biomass in 2017 relative to the treatment not harvested for forage. Soil indicators of carbon and nitrogen storage were not affected by forage harvest but mineralizable carbon, an indicator of nutrient cycling, was greater under the forage harvested treatments. The harvest of forage and grain promoted nutrient availability and overall productivity (forage, root and grain biomass) relative to harvesting for grain only. Our findings suggest dual-use management of Kernza can provide a productive and profitable pathway for perennial grain adoption.

In an era of constrained and depleted natural resources, perennial grains could provide sustainable food production along with beneficial ecosystem services like reduced erosion and increased atmospheric carbon capture. Intermediate wheatgrass (IWG) [Thinopyrum intermedium (Host) Barkworth & D. R. Dewey subsp. intermedium] has been undergoing continuous breeding for domestication to develop a perennial grain crop since the 1980s. As a perennial, IWG has required 2–5 yr per selection generation, but starting in 2017, genomic selection (GS) was initiated in the breeding program at The Land Institute, Salina, KS (TLI), enabling one complete cycle per year. For each cycle, ∼4,000 seedlings were profiled using genotyping‐by‐sequencing (GBS) and genomic estimated breeding values (GEBVs) were calculated. Selection based on GEBVs identified ∼100 individuals to advance as parents each generation, while validation populations of 1,000–1,200 genets for GS model training were also selected using the genomic relationship matrix to represent genetic diversity in each cycle. The selected parents were randomly intermated in a greenhouse crossing block to form the subsequent cycle, while the validation populations were transplanted to irrigated and nonirrigated field sites for phenotypic evaluations in the following years. For priority breeding traits of seed mass, free threshing, and nonshattering, correlations between predicted values and observed data were >.5. The realized selection differential ranged from 11–23% for selected traits, and the expected genetic gains for these traits, including spike yield, ranged from 6 to 14% per year. Genomic selection is a powerful tool to speed the domestication and development of IWG and other perennial crops with extended breeding timelines.

Thinopyrum intermedium (2n = 6x = 42, JJJSJSStSt) is one of the important resources for the wheat improvement. So far, a few Th. intermedium (Thi)-specific molecular markers have been reported, but the number is far from enough to meet the need of identifying alien fragments in wheat-Th. intermedium hybrids. In this study, 5,877,409 contigs were assembled using the Th. intermedium genotyping-by-sequencing (GBS) data. We obtained 5,452 non-redundant contigs containing mapped Thi-GBS markers with less than 20% similarity to the wheat genome and developed 2,019 sequence-tagged site (STS) molecular markers. Among the markers designed, 745 Thi-specific markers with amplification products in Th. intermedium but not in eight wheat landraces were further selected. The distribution of these markers in different homologous groups of Th. intermedium varied from 47 (7/12/28 on 6J/6St/6JS) to 183 (54/62/67 on 7J/7St/7JS). Furthermore, the effectiveness of these Thi-specific markers was verified using wheat-Th. intermedium partial amphidiploids, addition lines, substitution lines, and translocation lines. Markers developed in this study provide a convenient, rapid, reliable, and economical method for identifying Th. intermedium chromosomes in wheat. In addition, this set of Thi-specific markers can also be used to estimate genetic and physical locations of Th. intermedium chromatin in the introgression lines, thus providing valuable information for follow-up studies such as alien gene mining.

Previous work indicated that bran removal promotes network formation in breads prepared from intermediate wheatgrass (IWG) flour. However, refinement reduces yields as well as contents of nutritionally beneficial compounds such as fiber. This study evaluated xylanase pretreatment of IWG bran as a processing option to enhance the properties of bread made with half of the original bran content. Xylanase pretreatment did not affect stickiness but significantly reduced hardness and increased specific loaf volumes compared to negative (without xylanase) and positive controls (with xylanase but without pretreatment). However, the surface of breads with pretreated bran was uneven due to structural collapse during baking. Fewer but larger gas cells were present due to pretreatment. Addition of ascorbic acid modulated these effects, but did not prevent uneven surfaces. Accessible thiol concentrations were slightly but significantly increased by xylanase pretreatment, possibly due to a less compact crumb structure. Endogenous xylanases (apparent activity 0.46 and 5.81 XU/g in flour and bran, respectively) may have been activated during the pretreatment. Moreover, Triticum aestivum xylanase inhibitor activity was also detected (193 and 410 InU/g in flour and bran). Overall, xylanase pretreatment facilitates incorporation of IWG bran into breads, but more research is needed to improve bread appearance.

Owing to the excellent resistance to abiotic and biotic stress, Thionpyrum intermedium (2n = 6x = 42, JJJsJsStSt) and Thinopyrum ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs. Disomic substitution lines (DSLs) carrying one pair of alien chromosomes are valuable bridge materials for novel genes transmission. In this study, six wheat-Thinopyrum DSLs were derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), characterized by a sequential fluorescence in situ hybridization (FISH)-genome in situ hybridization (GISH), a multicolor GISH (mc-GISH), and an analysis of wheat 15K SNP array combined with molecular marker selection. ES-9 and ES-10 were two wheat- Th. ponticum disomic substitution lines, DS2St (2A) and DS3St (3D). While ES-23, ES-24, ES-25, and ES-26 were four wheat- Th. intermedium disomic substitution lines, DS2St (2A), DS3St (3D), DS2St (2B), DS2St (2D). The FISH karyotypes of Th. ponticum 2St/3St chromosomes were well coincident with the ones of Th. intermedium. The chromosome configurations of F1 hybrids derived from crosses between ES-23 and ES-9, as well as ES-24 and ES-10 were mostly formed 21Ⅱ. Four St-chromosome-specific markers were developed by specific-locus amplified fragment sequencing (SLAF-seq). Additionally, the substitution lines containing chromosome 2St conferred higher thousand-kernel weight and stripe rust resistance at adult stages, while the substitution lines containing chromosome 3St were highly resistant to stripe rust at all stages. Therefore, these six substitution lines could serve as useful bridging parents for wheat genetic improvement.

The cultivation of perennial wheat might represent an economically and ecologically interesting option for extensive cultivation, particularly in marginal land. In autumn 2017, five breeding lines of perennial wheat (Triticum aestivum × Thinopyrum intermedium) and for comparison two varieties of annual wheat were sown at three sites in Bavaria. The five breeding lines were selected from a bulk originating from Washington State University. In 2018, the yield of the perennial breeding lines was 49 to 96 % of the annual cv. ‘Capo’, which reached an average of 17.4 dt/ha. In 2019, the yield was 9 to 38 % of ‘Capo’, which reached an average of 10.8 dt/ha, showing large differences between the locations. The autumn re-emergence was significantly affected by the severe drought at all sites in 2018 and 2019 and showed differences between the lines up to a total failure at one site. In the third year, the yield of the breeding lines was very low, thus a third year of cultivation cannot be recommended under these climatic and soil conditions. The yields of the plots with under-sown clover were significantly higher in the third year. A mixture with a low growing clover species is recommended because of better weed suppression. Field trials in Austria with perennial rye cv. ‘Perenne’ derived from an interspecific cross between Secale cereale and S. montanum were carried out from 2012 to 2015 in order to determine biomass yield and regrowth capability. Biomass yield of ‘Perenne’ in the early cuttings was between 70 and 95% compared to cv. ‘Elego’, but decreased to 45% after heading/anthesis for the two- and three-year crop stand. This decrease was mainly due to the reduced plant height (-45 cm) of the perennial crop in the 2nd and 3rd year compared to the first year crop. Root mass in the three-year crop was significantly higher compared to ‘Elego’ and the first and second year ‘Perenne’, but mainly only in the top 20 cm. Generally, significantly inferior grain yield was obtained for ‘Perenne’, reaching only 53%, 26% and 15% for the 1st, 2nd and 3rd year crop, respectively, compared to annual ‘Elego’ (5670 kg/ha). Similarly, a significant decrease was also observed for thousand grain weight (-32%) from the first to the third year of cultivation. Moreover, a higher number of ergot sclerotia was observed for the interspecific perennial rye hybrid compared to ‘Elego’. Weed infestation was constantly increasing in the perennial crop making a cultivation of more than 3 years not recommendable.

Perennial grain crops have the potential to improve agricultural sustainability but few existing species produce sufficient grain yield to be economically viable. The outcrossing, allohexaploid, and perennial forage species intermediate wheatgrass (IWG) [Thinopyrum intermedium (Host) Barkworth & D. R. Dewey] has shown promise in undergoing direct domestication as a perennial grain crop using phenotypic and genomic selection. However, decades of selection will be required to achieve yields on par with annual small‐grain crops. Marker‐aided selection could accelerate progress if important genomic regions associated with domestication were identified. Here we use the IWG nested association mapping (NAM) population, with 1,168 F1 progeny across 10 families to dissect the genetic control of brittle rachis, floret shattering, and threshability. We used a genome‐wide association study (GWAS) with 8,003 single nucleotide polymorphism (SNP) markers and linkage mapping—both within‐family and combined across families—with a robust phenotypic dataset collected from four unique year‐by‐location combinations. A total of 29 quantitative trait loci (QTL) using GWAS and 20 using the combined linkage analysis were detected, and most large‐effect QTL were in common across the two analysis methods. We reveal that the genetic control of these traits in IWG is complex, with significant QTL across multiple chromosomes, sometimes within and across homoeologous groups and effects that vary depending on the family. In some cases, these QTL align within 216 bp to 31 Mbp of BLAST hits for known domestication genes in related species and may serve as precise targets of selection and directions for further study to advance the domestication of IWG.

Thinopyrum intermedium (2n = 6x = 42, JJJSJSStSt) is one of the important resources for the wheat improvement. So far, a few Th. intermedium (Thi)-specific molecular markers have been reported, but the number is far from enough to meet the need of identifying alien fragments in wheat-Th. intermedium hybrids. In this study, 5,877,409 contigs were assembled using the Th. intermedium genotyping-by-sequencing (GBS) data. We obtained 5,452 non-redundant contigs containing mapped Thi-GBS markers with less than 20% similarity to the wheat genome and developed 2,019 sequence-tagged site (STS) molecular markers. Among the markers designed, 745 Thi-specific markers with amplification products in Th. intermedium but not in eight wheat landraces were further selected. The distribution of these markers in different homologous groups of Th. intermedium varied from 47 (7/12/28 on 6J/6St/6JS) to 183 (54/62/67 on 7J/7St/7JS). Furthermore, the effectiveness of these Thi-specific markers was verified using wheat-Th. intermedium partial amphidiploids, addition lines, substitution lines, and translocation lines. Markers developed in this study provide a convenient, rapid, reliable, and economical method for identifying Th. intermedium chromosomes in wheat. In addition, this set of Thi-specific markers can also be used to estimate genetic and physical locations of Th. intermedium chromatin in the introgression lines, thus providing valuable information for follow-up studies such as alien gene mining.

Background Thinopyrum intermedium (2n = 6x = 42) is an important wild perennial Triticeae species exhibiting many potentially favorable traits for wheat improvement. Wheat-Th. intermedium partial amphiploids serve as a bridge to transfer desirable genes from Th. intermedium into common wheat. Results Three octoploid Trititrigia accessions (TE261–1, TE266–1, and TE346–1) with good resistances to stripe rust, powdery mildew and aphids were selected from hybrid progenies between Th. intermedium and the common wheat variety ‘Yannong 15’ (YN15). Genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and multicolor GISH (McGISH) analyses demonstrated that the three octoploid Trititrigia possess 42 wheat chromosomes and 14 Th. intermedium chromosomes. The 14 alien (Th. intermedium) chromosomes belong to a mixed genome consisting of J-, JS- and St-genome chromosomes rather than a single J, JS or St genome. Different types of chromosomal structural variation were also detected in the 1A, 6A, 6B, 2D and 7D chromosomes via FISH, McGISH and molecular marker analysis. The identity of the alien chromosomes and the variationes in the wheat chromosomes in the three Trititrigia octoploids were also different. Conclusions The wheat-Th. intermedium partial amphiploids possess 14 alien chromosomes which belong to a mixed genome consisting of J-, JS- and St- chromosomes, and 42 wheat chromosomes with different structural variations. These accessions could be used as genetic resources in wheat breeding for the transfer of disease and pest resistance genes from Th. intermedium to common wheat.

SummaryThe biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt) is a crucial factor causing reduction of global wheat production. Wild wheat relatives, e.g. Thinopyrum intermedium, is one of the wild-used parents in wheat disease-resistant breeding. From T. intermedium line, we identified the aspartic acid protein gene, TiAP1, which involved in resistance against Bgt. TiAP1 is a secreted protein that accumulates in large amounts at the infection sites of powdery mildew and extends to the intercellular space. Yeast two-hybrid showed that it interacted with the chitin deacetylase (BgtCDA1) of Bgt. The yeast expression, purification, and invitro test confirmed the chitin deacetylase activity of BgtCDA1. The bombardment and VIGS mediated host-induced gene silencing showed BgtCDA1 promotes the invasion of Bgt. Transcriptome analysis showed the cell wall xylan metabolism, lignin biosynthesis-related, and defence genes involved in the signal transduction were upregulated in the transgenic TiAP1 wheat induced by Bgt. The TiAP1 in wheat may inactivate the deacetylation function of BgtCDA1, cause chitin oligomers expose to wheat chitin receptor, then trigger the wheat immune response to inhibit the growth and penetration of Bgt, and thereby enhance the tolerance of wheat to pathogens.

Kernza intermediate wheatgrass (Thinopyrum intermedium) is the first commercially developed perennial grain crop in North America, with multiple environmental and economic benefits. One of the major challenges for adoption of this dual-use forage and grain crop is the decline in grain yield in subsequent harvest years. Post-harvest management practices (e.g., chopping, burning, chemical, and mechanical thinning) could reduce the intraspecific competition for light and maintain Kernza grain yields over time. We aimed to identify management practices that improve light penetration and propose a conceptual model to explain the mechanisms contributing to Kernza grain yield. We applied 10 management practices after the first Kernza grain harvest in a randomized complete block design experiment with three replications, at two different locations in Wisconsin, USA. Light penetration increased when post-harvest management practices were applied. Mechanical or chemical thinning had relatively lower lodging and increased yield components per row, but not per area due to a reduction in the number of productive rows. Threshed grain yield per area in the second year of Kernza was similar among the treatments despite the differences in vegetative biomass generated. Further research is needed to optimize management practices to maintain Kernza grain yield over time.

Intermediate wheatgrass (Thinopyrum intermedium) is an outcrossing, cool season grass species currently undergoing direct domestication as a perennial grain crop. Though many traits are selection targets, understanding the genetic architecture of those important for local adaptation may accelerate the domestication process. Nested association mapping (NAM) has proven useful in dissecting the genetic control of agronomic traits many crop species, but its utility in primarily outcrossing, perennial species has yet to be demonstrated. Here, we introduce an intermediate wheatgrass NAM population developed by crossing ten phenotypically divergent donor parents to an adapted common parent in a reciprocal manner, yielding 1,168 F1 progeny from 10 families. Using genotyping by sequencing, we identified 8,003 SNP markers and developed a population-specific consensus genetic map with 3,144 markers across 21 linkage groups. Using both genomewide association mapping and linkage mapping combined across and within families, we characterized the genetic control of flowering time. In the analysis of two measures of maturity across four separate environments, we detected as many as 75 significant QTL, many of which correspond to the same regions in both analysis methods across 11 chromosomes. The results demonstrate a complex genetic control that is variable across years, locations, traits, and within families. The methods were effective at detecting previously identified QTL, as well as new QTL that align closely to the well-characterized flowering time orthologs from barley, including Ppd-H1 and Constans. Our results demonstrate the utility of the NAM population for understanding the genetic control of flowering time and its potential for application to other traits of interest.

A physical map of Thinopyrum intermedium chromosome 7J was constructed using translocation mapping, and a new seedling purple coleoptile gene was mapped to the bin of FL 0.35–0.63 of 7JS. Thinopyrum intermedium (2n = 6x = 42, JJJsJsStSt), a wild relative of common wheat, harbors numerous beneficial genes for wheat improvement. Previous studies showed that wheat–Th. intermedium partial amphiploid TAF46 and its derived addition line L1 had a purple coleoptile, which was derived from Th. intermedium chromosome 7J. To identify and physically map the purple coleoptile gene, 12 wheat–Th. intermedium 7J translocation lines were analyzed by sequential multicolor fluorescence in situ hybridization (mc-FISH), PCR-based landmark unique gene (PLUG) and intron targeting (IT) markers. A physical map of the 7J chromosome was constructed, consisting of eight chromosomal bins with 89 markers. Seedling evaluation of the coleoptile colors of all tested materials indicated that the purple coleoptile gene was located to the bin with a fraction length (FL) of 0.35–0.63 on chromosome 7JS. Furthermore, based on the syntenic relationships between Th. intermedium and wheat chromosomes, we developed a new chromosome 7J-specific EST-PCR marker from the chromosomal region corresponding to the purple coleoptile gene through the Triticeae multi-omics database. The approach of designing chromosome-specific markers has facilitated fine mapping of the Thinopyrum-specific purple coleoptile gene, and these translocation lines will be valuable for studying the function of the purple coleoptile gene in anthocyanin biosynthesis.

Perennial wheat is considered to be a practical way to increase the flexibility and profitability of sustainable agricultural system, as it can be either a forage grass or a grain crop. Four perennial wheat lines SX12-480, SX12-787, SX12-1150, and SX12-1269 were developed from a series of interspecific crosses between common wheat (Triticum aestivum, 2n = 42) or durum wheat (T. turgidum var. durum, 2n = 28) and the intermediate wheatgrass (Thinopyrum intermedium, 2n = 42). These lines were characterized by the vigorous regrowth for at least three years. The one- and two-year-old plants had higher grain yield potential than the three-year-old perennial plants. The decline of grain yield was associated with plant age-related effects on yield components. The perennial wheat lines were all resistant to both Heterodera avenae and H. filipjevi, the two distinct cereal cyst nematode species that occur in China, except that line SX12-787 exhibited moderate resistance only to H. avenae. The dual-purpose perennial wheat lines were evaluated for quality values of both defoliated grass and harvested grains in the form of amino acid profile, mineral concentration, and contents of protein and fiber. Difference in the quality profile was observed between the perennial lines. These perennial lines had an overall improved quality levels over those of the perennial wheat control Montana-2 (T. turgidum × Th. intermedium) and the annual wheat cultivar Jinchun 9. The amplification profiles of the molecular markers provided molecular evidence for the introgression of alien chromatin. Genomic in situ hybridization detected 16, 14, 14, and 12 Th. intermedium chromosomes in lines SX12-480 (2n = 48), SX12-787 (2n = 56), SX12-1150 (2n = 56), and SX12-1269 (2n = 54), respectively, in addition to either 32 or the complete set of wheat chromosomes. The four perennial wheat-Th. intermedium lines described here provide valuable sources of perennial wheat for the dual-purpose application of both grain and forage.

Perennial wheat is considered to be a practical way to increase the flexibility and profitability of sustainable agricultural system, as it can be either a forage grass or a grain crop. Four perennial wheat lines SX12-480, SX12-787, SX12-1150, and SX12-1269 were developed from a series of interspecific crosses between common wheat (Triticum aestivum, 2n = 42) or durum wheat (T. turgidum var. durum, 2n = 28) and the intermediate wheatgrass (Thinopyrum intermedium, 2n = 42). These lines were characterized by the vigorous regrowth for at least 3 years. The one- and 2-year-old plants had higher grain yield potential than the 3-year-old perennial plants. The decline of grain yield was associated with plant age-related effects on yield components. The perennial wheat lines were all resistant to both Heterodera avenae and H. filipjevi, the two distinct cereal cyst nematode species that occur in China, except that line SX12-787 exhibited moderate resistance only to H. avenae. The dual-purpose perennial wheat lines were evaluated for quality values of both defoliated grass and harvested grains in the form of amino acid profile, mineral concentration, and contents of protein and fiber. Difference in the quality profile was observed between the perennial lines. These perennial lines had an overall improved quality levels over those of the perennial wheat control Montana-2 (T. turgidum × Th. intermedium) and the annual wheat cultivar Jinchun 9. The amplification profiles of the molecular markers provided molecular evidence for the introgression of alien chromatin. Genomic in situ hybridization detected 16, 14, 14, and 12 Th. intermedium chromosomes in lines SX12-480 (2n = 48), SX12-787 (2n = 56), SX12-1150 (2n = 56), and SX12-1269 (2n = 54), respectively, in addition to either 32 or the complete set of wheat chromosomes. The four perennial wheat-Th. intermedium lines described here provide valuable sources of perennial wheat for the dual-purpose application of both grain and forage.

The development of perennial grain crops is driven by the vision of simultaneous food production and enhanced ecosystem services. Typically, perennial crops like intermediate wheatgrass (IWG)[Thinopyrum intermedium (Host) Barkworth & D.R Dewey] have low seed yield and other detrimental traits. Next‐generation sequencing has made genomic selection (GS) a tractable and viable breeding method. To investigate how an IWG breeding program may use GS, we evaluated 3,658 genets over 2 yr for 46 traits to build a training population. Six statistical models were used to evaluate the non‐replicated data, and a model using autoregressive order 1 (AR1) spatial correction for rows and columns combined with the genomic relationship matrix provided the highest estimates of heritability. Genomic selection models were built from 18,357 single nucleotide polymorphism markers via genotyping‐by‐sequencing, and a 20‐fold cross‐validation showed high predictive ability for all traits (r > .80). Predictive abilities improved with increased training population size and marker numbers, even with larger amounts of missing data per marker. On the basis of these results, we propose a GS breeding method that is capable of completing one cycle per year compared with a minimum of 2 yr per cycle with phenotypic selection. We estimate that this breeding approach can increase the rate of genetic gain up to 2.6× above phenotypic selection for spike yield in IWG, allowing GS to enable rapid domestication and improvement of this crop. These breeding methods should be transferable to other species with similar long breeding cycles or limited capacity for replicated observations.

Starch, as the main component of grain in cereals, serves as the major source of calories in staple food and as a raw material for industry. As the technological and digestive properties of starch depend on its content, the management of its components, amylose and amylopectin, is of great importance. The starch properties of wheat grain can be attuned using allelic variations of genes, including granule-bound starch synthase I (GBSS I), or Wx. The tertiary gene pool, including wheatgrass (Thinopyrum) species, provides a wide spectrum of genes-orthologs that can be used to increase the allelic diversity of wheat genes by wide hybridization. Octaploid partial wheat&ndash, wheatgrass hybrids (amphidiploids, WWGHs) combine the complete genome of bread wheat (BBAADD), and a mixed genome from the chromosomes of intermediate wheatgrass (Thinopyrum intermedium, genomic composition JrJrJvsJvsStSt) and tall wheatgrass (Th. ponticum, JJJJJJJsJsJsJs). Thus, WWGHs may carry Wx genes not only of wheat (Wx-B1, Wx-A1 and Wx-D1) but also of wheatgrass origin. We aimed to assess the level of amylose in starch and investigate the polymorphism of Wx genes in 12 accessions of WWGHs. Additionally, we characterized orthologous Wx genes in the genomes of wild wheat-related species involved in the development of the studied WWGHs, Th. intermedium and Th. ponticum, as well as in the putative donors of their subgenomes, bessarabian wheatgrass (Th. bessarabicum, JbJb) and bluebunch wheatgrass (Pseudoroegneria stipifolia, St1St1St2St2). Although no significant differences in amylose content were found between different WWGH accessions, SDS-PAGE demonstrated that at least two WWGHs have an additional band. We sequenced the Wx gene-orthologs in Th. bessarabicum, P. stipifolia, Th. intermedium and Th. ponticum, and developed a WXTH marker that can discriminate the Thinopyrum Wx gene in the wheat background, and localized it to the 7E chromosome in Th. elongatum. Using the WXTH marker we revealed the allelic polymorphism of the Thinopyrum Wx gene in the studied WWGHs. The applicability of Thinopyrum Wx genes in wheat breeding and their effect on starch quality are discussed.

The cytogenetic study of wide hybrids of wheat has both practical and fundamental values. Partial wheat-wheatgrass hybrids (WWGHs) are interesting as a breeding bridge to confer valuable genes to wheat genome, as well as a model object that contains related genomes of Triticeae. The development of cytogenetic markers is a process that requires long and laborious fluorescence in situ hybridization (FISH) testing of various probes before a suitable probe is found. In this study, we aimed to find an approach that allows to facilitate this process. Based on the data sequencing of Thinopyrum ponticum, we selected six tandem repeat (TR) clusters using RepeatExplorer2 pipeline and designed primers for each of them. We estimated the found TRs&rsquo, abundance in the genomes of Triticum aestivum, Thinopyrum ponticum, Thinopyrum intermedium and four different WWGH accessions using real-time qPCR, and localized them on the chromosomes of the studied WWGHs using fluorescence in situ hybridization. As a result, we obtained three tandem repeat cytogenetic markers that specifically labeled wheatgrass chromosomes in the presence of bread wheat chromosomes. Moreover, we designed and tested primers for these repeats, and demonstrated that they can be used as qPCR markers for quick and cheap monitoring of the presence of certain chromosomes of wheatgrass in breeding programs.

Cold-hardy perennial wheatgrass plays an important role in the use of barren land for farming, soil and water conservation, variety improvement, and also for increasing grass yield. By crossing octoploid tritelytrigia (2n = 8x = 56, AABBDDEE) with Thinopyrum intermedium (2n = 6x = 42, StStJJJ S J S ), we developed 34 lines of perennial wheatgrass from F1 to F6 generations, which had vigorous regrowth and cold hardiness. The cold-hardy, perennial wheatgrass lines were well-adapted to the cold environment and developed root and rhizomes, with a longevity between 5 and 11 years and a better seed set. Some of them maintained wheat chromosomes beneficial for breeding perennial wheat. Molecular cytogenetic analysis demonstrated that the Th. intermedium chromosomes contributed the most to the synthetic genome of the wheatgrass hybrids and were associated with the perennial growth habit and winter hardiness. They were also preferentially maintained and transmitted to the progenies. Some wheat chromosomes were also transmitted from the F1 to F6 generations, although they were eliminated in each life cycle of the wheatgrass hybrids. The numbers of wheat and Th. intermedium chromosomes affected seed set and perennial growth habit. Seed set increased with the establishment of a more balanced genomic constitution in later generations. The cold-hardy and perennial wheatgrass lines were produced, which can be the starting point of domestication effort aimed at producing well-adapted ground cover plants under extreme environments.