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Abstract Background Developing novel germplasm by using wheat wild related species is an effective way to rebuild the wheat resource bank. The Psathyrostachys huashanica Keng (P. huashanica, 2n = 2x = 14, NsNs) is regarded as a superior species to improve wheat breeding because of its multi-resistance, early maturation and numerous tiller traits. Introducing genetic components of P. huashanica into the common wheat background is the most important step in achieving the effective use. Therefore, the cytogenetic characterization and influence of the introgressed P. huashanica large segment chromosomes in the wheat background is necessary to be explored. Results In this study, we characterized a novel derived line, named D88-2a, a progeny of the former characterized wheat-P. huashanica partial amphiploid line H8911 (2n = 7x = 49, AABBDDNs). Cytological identification showed that the chromosomal composition of D88-2a was 2n = 44 = 22II, indicating the addition of exogenous chromosomes. Genomic in situ hybridization demonstrated that the supernumerary chromosomes were a pair of homologues from the P. huashanica and could be stably inherited in the common wheat background. Molecular markers and 15 K SNP array indicated that the additional chromosomes were derived from the sixth homoeologous group (i.e., 6Ns) of P. huashanica. Based on the distribution of the heterozygous single-nucleotide polymorphism sites and fluorescence in situ hybridization karyotype of each chromosome, this pair of additional chromosomes was confirmed as P. huashanica 6Ns large segment chromosomes, which contained the entire short arm and the proximal centromere portion of the long arm. In terms of the agronomic traits, the addition line D88-2a exhibited enhanced stripe rust resistance, improved spike characteristics and increased protein content than its wheat parent line 7182. Conclusions The new wheat germplasm D88-2a is a novel cytogenetically stable wheat-P. huashanica 6Ns large segment addition line, and the introgressed large segment alien chromosome has positive impact on plant spikelet number and stripe rust resistance. Thus, this germplasm can be used for genetic improvement of cultivated wheat and the study of functional alien chromosome segment.

Akebia trifoliata, as a new fruit, is becoming competitive and popular in the markets of eastern Asian countries, especially China, because of its nutritional value and health-promoting functions. To ultimately develop seedless varieties, germinating seeds of the A. trifoliata monoembryonic line ‘710’ with ∼1.5-cm-long roots were treated with five different concentrations of colchicine (0.1%, 0.2%, 0.3%, 0.4%, and 0.5%) for four soaking periods (12, 24, 36, and 48 hours). Ploidy level assessments via both flow cytometry and karyotype analysis revealed some autotetraploids and chimeras in surviving seedlings treated with both 0.4% and 0.5% colchicine for 48 h, but the highest autotetraploid (33.3%) and chimera (19.0%) rates were observed in surviving seedlings treated with 0.3% colchicine for a 12-hour soaking period; no autotetraploids or chimeras were detected in plants treated with only water. In addition, we also found that autotetraploid plants usually presented broader and thicker leaves with larger stomas and epidermic cortical cells. Notably, no autotetraploid of A. trifoliata has been reported previously; therefore, both autotetraploids and chimeras are valuable breeding parents for autotriploid seedless varieties and ideal materials for further theoretical studies.

The wild species Thinopyrum intermedium (genome JJJSJSStSt) serves as a valuable germplasm resource providing novel diseases resistance and agronomically important genes for wheat improvement. Two wheat-Th. intermedium partial amphiploids, TAI7045 (2n = 56) and 78784 (2n = 56), exhibit high resistance to stripe rust and powdery mildew, and their chromosome constitutions have been characterized. With the aim to transfer novel resistance genes from Th. intermedium, the crosses of common wheat line MY11 with TAI7045 and 78784 were produced, and their individual F2-F5 progenies were characterized using sequential non-denaturing fluorescence in situ hybridization (ND-FISH) and molecular markers. We identified a set of wheat-Th. intermedium addition lines, involving the chromosomes 1St-JS, 2St, 2St-JS, 3St, 4J, 4St, 5St, 5J.St, 6JS.J, and 7JS. Above all, the stable wheat-Th. intermedium small segmental translocation lines with chromosomes 4DS.4DL-4StL-4DL-4JL and 4DS.4DL-4StL-4DL were selected. Combining data from specific marker amplification and resistance evaluation, we mapped the gene(s) for resistance to powdery mildew and stripe rust in the 233.56–329.88 Mb region of the long arm of the 4St chromosome from the reference Th. intermedium genome. The new wheat-Th. intermedium introgressions will be used as novel germplasm for breeding purposes.

Genetic transformation is widely used to improve target traits and to study gene function in wheat. However, transformation efficiency depends on the physiological status of the recipient genotype and that is affected by several factors including powdery mildew (PM) infection. The widely used recipient variety Fielder is very susceptible to PM. Therefore, it would be beneficial to develop PM resistant derivatives with high regeneration ability for use in genetic transformation. In the present study PM resistant lines CB037 and Pm97033 carrying genes Pm21 and PmV, respectively, were backcrossed to Fielder with selection for PM resistance. Five lines, NT89, NT90, NT154, and WT48 with Pm21 and line FL347 with PmV were developed, identified by molecular markers and genomic in situ hybridization (GISH) or fluorescent in situ hybridization (FISH), and further subjected to detailed assessment of agronomic traits and regeneration ability following genetic transformation capacity. Lines FL347, WT48, NT89 and NT154 assessed as being equal to, or superior, to Fielder in regeneration and transformation ability are recommended as suitable materials for the replacement of Fielder for wheat gene transfer and genome editing study.

Polyploidization plays important roles in the evolution and breeding of the common wheat. Aegilops tauschii, the D-genome progenitor of the common wheat, provides a valuable pool of resistance genes to multiple diseases. Extensive studies focus on the exploration of these genes for wheat improvement. However, few studies have unveiled alternations on genome-wide expression pattern and histone modifications induced by whole-genome doubling (WGD) process. In this study, we conducted transcriptome analysis for the diploid and tetraploid Ae. taushcii lines using the leaf and root tissues. Both lines tend to display similar tissue-specific pattern. Interestingly, we found that TEs located in genic regions were depleted of the repressive histone mark H3K27me3, whereas their adjacent chromatin was enriched with H3K27me3. The tetraploid line exhibited higher levels of H3K27me3 in those regions than the diploid line, particularly for genic regions associated with TEs of the long interspersed nuclear elements (LINEs), CACTA, PIF/Harbinger, Tc1/Mariner and unclassed DNA transposon. Surprisingly, the expression levels of these TEs cognate genes were negatively associated with the levels of H3K27me3 between the tetraploid and diploid lines, suggesting the five types of TEs located within genic regions might be involved in the regulation of the ploidy-related gene expression, possibly through differential enrichment of H3K27me3 in the genic regions. These findings will help to understand the potential role of specific types of TEs on transcription in response to WGD.

An elite hexaploid triticale Yukuri from Australia was used as a bridge for transferring valuable genes from Secale cereale L. into common wheat for enriching the genetic variability of cultivated wheat. Non-denaturing-fluorescence in situ hybridization (ND-FISH) identified that Yukuri was a secondary triticale with a complete set of rye chromosomes and a 6D(6A) substitution. Seed protein electrophoresis showed that Yukuri had a unique composition of glutenin subunits. A set of Yukuri-derived wheat-rye introgression lines were created from a Yukuri x wheat population, and all lines were identified by ND-FISH with multiple probes and validated by diagnostic molecular marker analysis. A total of 59 wheat-rye introgression lines including modified chromosome structural variations of wheat, and new complex recombinant chromosomes of rye were detected through ND-FISH and Oligo-FISH painting based on oligonucleotide pools derived from wheat-barley genome collinear regions. Wheat lines carrying the 1R chromosome from Yukuri displayed resistance to both stripe rust and powdery mildew, while the lines carrying the 3RL and 7RL chromosome arms showed stripe rust resistance. The chromosome 1R-derived lines were found to exhibit a significant effect on most of the dough-related parameters, and chromosome 5R was clearly associated with increased grain weight. The development of the wheat-rye cytogenetic stocks carrying disease resistances and superior agronomic traits, as well as the molecular markers and FISH probes will promote the introgression of abundant variation from rye into wheat improvement programs.

As a perennial herb in Triticeae, Elymus dahuricus is widely distributed in Qinghai–Tibetan Plateau and Central Asia. It has been used as high-quality fodders for improving degraded grassland. The genomic constitution of E. dahuricus (2n = 6x = 42) has been revealed as StStHHYY by cytological approaches. However, the universal karyotyping nomenclature system of E. dahuricus is not fully established by traditional fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH). In this study, the non-denaturing fluorescent in situ hybridization (ND-FISH) using 14 tandem-repeat oligos could effectively distinguish the entire E. dahuricus chromosomes pairs, while Oligo-FISH painting by bulked oligo pools based on wheat-barley collinear regions combined with GISH analysis, is able to precisely determine the linkage group and sub-genomes of the individual E. dahuricus chromosomes. We subsequently established the 42-chromosome karyotype of E. dahuricus with distinctive chromosomal FISH signals, and characterized a new type of intergenomic rearrangement between 2H and 5Y. Furthermore, the comparative chromosomal localization of the centromeric tandem repeats and immunostaining by anti-CENH3 between cultivated barley (Hordeum vulgare L.) and E. dahuricus suggests that centromere-associated sequences in H subgenomes were continuously changing during the process of polyploidization. The precise karyotyping system based on ND-FISH and Oligo-FISH painting methods will be efficient for describing chromosomal rearrangements and evolutionary networks for polyploid Elymus and their related species.

Rye 6R-derived stripe rust resistance gene Yr83 in wheat background was physically mapped to fraction length (FL) 0.87-1.00 on the long arm by non-denaturing-fluorescence in situ hybridization (ND-FISH), Oligo-FISH painting and 6R-specific PCR markers.Stripe rust resistance gene Yr83 derived from chromosome 6R of rye (Secale cereale) “Merced” has displayed high resistance to both Australian and Chinese wheat stripe rust isolates. With the aim to physically map Yr83 to a more precise region, new wheat- 6R deletion and translocation lines were produced from derived progenies of the 6R(6D) substitution line. The non-denaturing fluorescence in situ hybridization (ND-FISH) patterns of 6R were established to precisely characterize the variations of 6R in different wheat backgrounds. Comparative ND-FISH analysis localized the breakpoints of 6RL chromosomes relative to Oligo-pSc200 and Oligo-pSc119.2 rich sites in deletion lines. Molecular marker and resistance analyses confirmed that Yr83 is physically located at the fraction length (FL) 0.87-1.00 of 6RL and covers the corresponding region of 806-881 Mb in the reference genome of Lo7. Oligo-FISH painting demonstrated that the region carrying Yr83 is syntenic to the distal end of long arm of homoeologous group 7 of the Triticeae genome. The developed wheat-6R lines carrying the Yr83 gene will be useful for breeding for rust resistance.

It was reported that the chromosome 6R of rye (Secale cereale L.) carries stripe rust resistance gene Yr83, and the region with the candidate resistance gene(s) still needs to be narrowed down. This study confirmed that the chromosome 6RLAr derived from rye AR106BONE contains stripe rust resistance gene(s). A wheat-rye T6BS.6RLAr translocation chromosome, a wheat-rye small-segment translocation T6RLAr-6AS.6AL, and three kinds of deleted T6BS.6RLAr translocations, T6BS.6RLAr-1, T6BS.6RLAr-2, and T6BS.6RLAr-3, were identified. Translocations T6BS.6RLAr, T6BS.6RLAr-2, and T6RLAr-6AS.6AL were highly resistant to stripe rust and T6BS.6RLAr-1 and T6BS.6RLAr-3 were highly susceptible. The molecular markers specific to 6RL determined that the three regions of the 6RLAr arm from 732,999,830 bp to the telomere, from 735,010,030 to 848,010,414 bp, and from 848,011,262 bp to the telomere were deleted from T6BS.6RLAr-1, T6BS.6RLAr-2, and T6BS.6RLAr-3, respectively. T6BS.6RLAr-2 and T6RLAr-6AS.6AL contained the segment that was deleted in T6BS.6RLAr-3. Therefore, it can be concluded that about 37 Mb segment from 848,011,262 bp to the telomere carried stripe rust resistance gene(s), and it was smaller than that with the Yr83 gene. Gene annotation indicated that about 37 Mb region contains 43 potential resistance genes, and 42 of them are nucleotide-binding site and leucine-rich repeat (NBS-LRR)-like resistance protein genes. The results in this study narrowed down the size of the region with candidate stripe rust resistance gene(s) on the 6RL arm, and the T6RLAr-6AS.6AL is a promising small-segment translocation for improvement of wheat cultivars.

Abstract Background The identification of chromosomes among Avena species have been studied by C-banding and in situ hybridization. However, the complicated results from several cytogenetic nomenclatures for identifying oat chromosomes are often contradictory. A universal karyotyping nomenclature system for precise chromosome identification and comparative evolutionary studies would be essential for genus Avena based on the recently released genome sequences of hexaploid and diploid Avena species. Results Tandem repetitive sequences were predicted and physically located on chromosomal regions of the released Avena sativa OT3098 genome assembly v1. Eight new oligonucleotide (oligo) probes for sequential fluorescence in situ hybridization (FISH) were designed and then applied for chromosome karyotyping on mitotic metaphase spreads of A. brevis, A. nuda, A. wiestii, A. ventricosa, A. fatua, and A. sativa species. We established a high-resolution standard karyotype of A. sativa based on the distinct FISH signals of multiple oligo probes. FISH painting with bulked oligos, based on wheat-barley collinear regions, was used to validate the linkage group assignment for individual A. sativa chromosomes. We integrated our new Oligo-FISH based karyotype system with earlier karyotype nomenclatures through sequential C-banding and FISH methods, then subsequently determined the precise breakage points of some chromosome translocations in A. sativa. Conclusions This new universal chromosome identification system will be a powerful tool for describing the genetic diversity, chromosomal rearrangements and evolutionary relationships among Avena species by comparative cytogenetic and genomic approaches.

The number of spikelets per spike is an important trait that directly affects grain yield in wheat. Three quantitative trait loci (QTLs) associated with spikelet nodes per spike (SNS) were mapped in a population of recombinant inbred lines generated from a cross between two advanced breeding lines of winter wheat based on the phenotypic variation evaluated over six locations/years. Two of the three QTLs are QSns.sxau-2A at the WHEATFRIZZY PANICLE (WFZP) loci and QSns.sxau-7A at the WHEAT ORTHOLOG OF APO1 (WAPO1) loci. The WFZP-A1b allele with a 14-bp deletion at QSns.sxau-2A was associated with increased spikelets per spike. WAPO-A1e, as a novel allele at WAPO1, were regulated at the transcript level that was associated with the SNS trait. The third SNS QTL, QSns.sxau-7D on chromosome 7D, was not associated with homoeologous WAPO-D1 or any other genes known to regulate SNS. The favorable alleles for each of WZFP-A1, WAPO-A1, and QSns.sxau-7D are identified and incorporated to increase up to 3.4 spikelets per spike in the RIL lines. Molecular markers for the alleles were developed. This study has advanced our understanding of the genetic basis of natural variation in spikelet development in wheat.

Even frequently used in wheat breeding, we still have an insufficient understanding of the biology of the products via distant hybridization. In this study, a transcriptomic analysis was performed for six Triticum aestivum-Thinopyrum elongatum substitution lines in comparison with the host plants. All the six disomic substitution lines showed much stronger “transcriptomic-shock” occurred on alien genomes with 57.43–69.22% genes changed expression level but less on the recipient genome (2.19–8.97%). Genome-wide suppression of alien genes along chromosomes was observed with a high proportion of downregulated genes (39.69–48.21%). Oppositely, the wheat recipient showed genome-wide compensation with more upregulated genes, occurring on all chromosomes but not limited to the homeologous groups. Moreover, strong co-upregulation of the orthologs between wheat and Thinopyrum sub-genomes was enriched in photosynthesis with predicted chloroplastic localization, which indicates that the compensation happened not only on wheat host genomes but also on alien genomes.

Abstract Background Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) carries many outstanding agronomic traits, therefore is a valuable resource for wheat genetic improvement. Wheat–P. huashanica translocation lines are important intermediate materials for wheat breeding and studying the functions of alien chromosomes. However, powdery mildew resistance in these translocation lines has not been reported previously. Results This study developed a novel wheat–P. huashanica translocation line TR77 by selecting a F7 progeny from the cross between heptaploid hybrid H8911 (2n = 7x = 49, AABBDDNs) and durum wheat line Trs-372. Chromosome karyotype of 2n = 42 = 21II was observed in both mitotic and meiotic stages of TR77. Genomic in situ hybridization analysis identified two translocated chromosomes that paired normally at meiosis stage in TR77. Molecular marker analysis showed that part of chromosome 5D was replaced by part of alien chromosome fragment 5Ns. It meant replacement made part 5DL and part 5NsL·5NsS existed in wheat background, and then translocation happened between these chromosomes and wheat 3D chromosome. Fluorescence in situ hybridization demonstrated that TR77 carries dual translocations: T3DS-5NsL·5NsS and T5DL-3DS·3DL. Analysis using a 15 K-wheat-SNP chip confirmed that SNP genotypes on the 5D chromosome of TR77 matched well with these of P. huashanica, but poorly with common wheat line 7182. The translocation was physically located between 202.3 and 213.1 Mb in 5D. TR77 showed longer spikes, more kernels per spike, and much better powdery mildew resistance than its wheat parents: common wheat line 7182 and durum wheat line Trs-372. Conclusions TR77 is a novel stable wheat–P. huashanica T3DS-5NsL·5NsS and T5DL-3DS·3DL dual translocation line and showed significant improved spike traits and resistance to powdery mildew compared to its parents, thus, it can be an useful germplasm for breeding disease resistance and studying the genetic mechanism of dual translocations.

Abstract Background Introgression of chromatin from Secale species into common wheat has for decades been a successful strategy for controlling the wheat diseases. The wild Secale species, Secale africanum Stapf., is a valuable source for resistance to foliar disease of wheat. A wheat-S. africanum chromosome 6Rafr substitution line displayed resistance to both powdery mildew and stripe rust at the adult-plant stage. Results Wheat-S. africanum chromosome 6Rafr deletion and translocation lines were produced and identified by sequential non-denaturing fluorescence in situ hybridization (ND-FISH) using multiple Oligo-based probes. Different ND-FISH patterns were observed between S. cereale 6R and S. africanum 6Rafr. With reference to the physical map of the draft genome sequence of rye inbred line Lo7, a comprehensive PCR marker analysis indicated that insertions and deletions had occurred by random exchange between chromosomes 6R and 6Rafr. A survey of the wheat- S. africanum 6Rafr lines for disease resistance indicated that a powdery mildew resistance gene(s) was present on the long arm of 6Rafr at FL0.85–1.00, and that a stripe rust resistance gene(s) was located in the terminal region of 6RafrS at FL0.95–1.00. The wheat-S. africanum 6Rafr introgression lines also displayed superior agronomic traits, indicating that the chromosome 6Rafr may have little linkage drag in the wheat background. Conclusions The combination of molecular and cytogenetic methods allowed to precisely identify the chromosome rearrangements in wheat- S. africanum 6Rafr substitution, deletion and translocation lines, and compare the structural difference between chromosomes 6R and 6Rafr. The wheat- S. africanum 6Rafr lines containing gene(s) for powdery mildew and stripe rust resistance could be used as novel germplasm for wheat breeding by chromosome engineering.

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

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.

Wheat (Triticum aestivum L.) is rich in tandem repeats, and this is helpful in studying its karyotypic evolution. Some tandem repeats have not been assembled into the wheat genome sequence. Alignment using the blastn tool in the B2DSC web server indicated that the genomic sequence of 5B chromosome (IWGSC RefSeq v2.1) does not contain the tandem repeat pTa-275, and the tandem repeat (GA)26 distributed throughout the whole 5B chromosome. The nondenaturing fluorescence in situ hybridization (ND-FISH) using the oligonucleotide (oligo) probes derived from pTa-275 and (GA)26 indicated that one signal band of pTa-275 and two signal bands of (GA)26 appeared on the 5B chromosome of Chinese Spring wheat, indicating the aggregative distribution patterns of the two kinds of tandem repeats. Single-copy FISH indicated that the clustering region of pTa-275 and the two clustering regions of (GA)26 were located in ~160–201 Mb, ~153–157 Mb, and ~201–234 Mb intervals, respectively. Using ND-FISH and single-copy FISH technologies, the translocation breakpoint on the 5BS portion of the translocation T7BS.7BL-5BS, which exists widely in north-western European wheat cultivars, was located in the region from 157,749,421 bp to 158,555,080 bp (~0.8 Mb), and this region mainly contains retrotransposons, and no gene was found. The clustering regions of two kinds of tandem repeats on wheat chromosome 5B were determined and this will be helpful to improve the future sequence assembly of this chromosome. The sequence characteristics of the translocation breakpoint on the translocation T7BS.7BL-5BS obtained in this study are helpful to understand the mechanism of wheat chromosome translocation.

Aegilops sharonensis, a wild relative of wheat, harbors diverse disease and insect resistance genes, making it a potentially excellent gene source for wheat improvement. In this study, we characterized and evaluated six wheat-A. sharonensis derivatives, which included three disomic additions, one disomic substitution + monotelosomic addition and two disomic substitution + disomic additions. A total of 51 PLUG markers were developed and used to allocate the A. sharonensis chromosomes in each of the six derivatives to Triticeae homoeologous groups. A set of cytogenetic markers specific for A. sharonensis chromosomes was established based on FISH using oligonucleotides as probes. Molecular cytogenetic marker analysis confirmed that these lines were a CS-A. sharonensis 2Ssh disomic addition, a 4Ssh disomic addition, a 4Ssh (4D) substitution + 5SshL monotelosomic addition, a 6Ssh disomic addition, a 4Ssh (4D) substitution + 6Ssh disomic addition and a 4Ssh (4D) substitution + 7Ssh disomic addition line, respectively. Disease resistance investigations showed that chromosome 7Ssh of A. sharonensis might harbor a new powdery mildew resistance gene, and therefore it has potential for use as resistance source for wheat breeding.

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.

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.

The construction of the 28-chromosome karyotype of Dasypyrum breviaristatum was undertaken using multicolor non-denaturing fluorescent in situ hybridization (ND-FISH) and Oligo-FISH painting protocols. A novel wheat-D. breviaristatum line D2138 contained 44 chromosomes including a pair of D. breviaristatum 6VbS.2VbL translocation chromosomes. Individual F2 and F3 progenies of a cross between D2138 with wheat lines CM62, MY11 and JM22, respectively, were characterized using ND-FISH and molecular markers. A relatively high chromosome alteration rate within wheat and D. breviaristatum 6VbS and 2VbL was observed in the three progeny populations, suggesting that chromosome 6VbS.2VbL has a gametocidal-like gene. The different types of translocation and deletion lines allowed localization of D. breviaristatum-specific gliadin coding genes on sub-telomeric regions of 6VbS by PCR and acid polyacrylamide gel electrophoresis analysis. The positive effect of the D. breviaristatum 6VbS on agronomic and quality characters was also demonstrated. The new wheat-D. breviaristatum 6VbS and 2VbL translocation lines will be useful as novel germplasm for breeding purposes.

Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) and Leymus mollis Trin. (2n = 4x = 28, NsNsXmXm) are valuable resources for wheat breeding improvement as they share the Ns genome, which contains diverse resistance genes. To explore the behaviors and traits of Ns chromosomes from the two species in wheat background, a series of wheat–P. huashanica and wheat–L. mollis substitution lines were developed. In the present study, line DH109 (F7 progeny of wheat–P. huashanica heptaploid line H8911 × durum wheat Trs-372) and line DM131 (F8 progeny of wheat–L. mollis octoploid line M842 × durum wheat Trs-372) were selected. Cytological observation combined with genomic in situ hybridization experiments showed that DH109 and DM131 each had 20 pairs of wheat chromosomes plus a pair of alien chromosomes (Ns chromosome), and the pair of alien chromosomes showed stable inheritance. Multiple molecular markers and wheat 55K SNP array demonstrated that a pair of wheat 3D chromosome in DH109 and in DM131 was substituted by a pair of P. huashanica 3Ns chromosome and a pair of L. mollis 3Ns chromosome, respectively. Fluorescence in situ hybridization (FISH) analysis confirmed that wheat 3D chromosomes were absent from DH109 and DM131, and chromosomal FISH karyotypes of wheat 3D, P. huashanica 3Ns, and L. mollis 3Ns were different. Moreover, the two lines had many differences in agronomic traits. Comparing with their wheat parents, DH109 expressed superior resistance to powdery mildew and fusarium head blight, whereas DM131 had powdery mildew resistance, longer spike, and more tiller number. Therefore, Ns genome from P. huashanica and L. mollis might have some different effects. The two novel wheat–alien substitution lines provide new ideas and resources for disease resistance and high-yield breeding on further utilization of 3Ns chromosomes of P. huashanica or L. mollis.

Germplasm is the material basis for crop genetic improvement and related basic research. Knowledge of genetic diversity present in wheat is the prerequisite for wheat breeding and improvement. Non-denaturing fluorescence in situ hybridization (ND-FISH) is a powerful tool to distinguish chromosomal polymorphisms and evaluate genetic diversity in wheat. In this study, ND-FISH using Oligo-pSc119.2-1, Oligo-pTa535-1, and Oligo-(GAA)7 as probes were used to analyze the genetic diversity among 60 International Maize and Wheat Improvement Center (CIMMYT) derived wheat lines, and 93 cultivated wheat and landraces from the Chinese wheat core germplasm. A total of 137 polymorphic FISH patterns were obtained, in which 41, 65, and 31 were from A-, B-, and D-genome chromosomes, respectively, indicating polymorphism of B-genome > A-genome > D-genome. In addition, 22 and 51 specific FISH types were observed in the two germplasm resource lines. Twelve types of rearrangements, including seven new translocations, were detected in all 153 wheat lines. Genetic relationships among 153 wheat lines were clustered into six groups. Our research provides cytological information for rational utilization of wheat germplasm resources.

Mutations induced by radiation are widely used for developing new varieties of plants. To better understand the frequency and pattern of irradiation-induced chromosomal rearrangements, we irradiated the dry seeds of Chinese Spring (CS)-Dasypyrum villosum nullisomic-tetrasomic (6A/6D) addition (6V) line (2n = 44), WD14, with 60Co-γ-rays at dosages of 100, 200, and 300 Gy. The M0 and M1 generations were analyzed using Feulgen staining and non-denaturing fluorescence in situ hybridization (ND-FISH) by using oligonucleotide probes. Abnormal mitotic behavior and chromosomes with structural changes were observed in the M0 plants. In all, 39 M1 plants had structurally changed chromosomes, with the B genome showing the highest frequency of aberrations and tendency to recombine with chromosomes of the D genome. In addition, 19 M1 plants showed a variation in chromosome number. The frequency of chromosome loss was considerably higher for 6D than for the alien chromosome 6V, indicating that 6D is less stable after irradiation. Our findings suggested that the newly obtained γ-induced genetic materials might be beneficial for future wheat breeding programs and functional gene analyses.

Non-denaturing FISH (ND-FISH) technology has been widely used to study the chromosomes of Triticeae species because of its convenience. The oligo probes for ND-FISH analysis of wheat (Triticum aestivum L.) chromosomes are still limited. In this study, the whole genome shotgun assembly sequences (IWGSC WGA v0.4) and the first version of the reference sequences (IWGSC RefSeq v1.0) of Chinese Spring (T. aestivum L.) were used to find new tandem repeats. One hundred and twenty oligo probes were designed according to the new tandem repeats and used for ND-FISH analysis of chromosomes of wheat Chinese Spring. Twenty nine of the 120 oligo probes produce clear or strong signals on wheat chromosomes. Two of the 29 oligo probes can be used to conveniently distinguish wheat A-, B-, and D-genome chromosomes. Sixteen of the 29 oligo probes only produce clear or strong signals on the subtelomeric regions of 1AS, 5AS, 7AL, 4BS, 5BS, and 3DS arms, on the telomeric regions of 1AL, 5AL, 2BS, 3BL, 6DS, and 7DL arms, on the intercalary regions of 4AL and 2DL arms, and on the pericentromeric regions of 3DL and 6DS arms. Eleven of the 29 oligo probes generate distinct signal bands on several chromosomes and they are different from those previously reported. In addition, the short and long arms of 6D chromosome have been confirmed. The new oligo probes developed in this study are useful and convenient for distinguishing wheat chromosomes or specific segments of wheat chromosomes.

As an important relative of wheat (Triticum aestivum L), Dasypyrum breviaristatum contains novel high molecular weight glutenin subunits (HMW-GSs) encoded by Glu-1Vb genes. We identified new wheat—D. breviaristatum chromosome introgression lines including chromosomes 1Vb and 1VbL.5VbL by fluorescence in situ hybridization (FISH) combined with molecular markers. We found that chromosome changes occurred in the wheat—D. breviaristatum introgression lines and particularly induced the deletion of 5BS terminal repeats and formation of a new type of 5B-7B reciprocal translocation. The results imply that the D. breviaristatum chromosome 1Vb may contain genes which induce chromosomal recombination in wheat background. Ten putative high molecular weight glutenin subunit (HMW-GS) genes from D. breviaristatum and wheat—D. breviaristatum introgression lines were isolated. The lengths of the HMW-GS genes in Dasypyrum were significantly shorter than typical HMW-GS of common wheat. A new y-type HMW-GS gene, named Glu-Vb1y, was characterized in wheat—D. breviaristatum 1Vb introgression lines. The new wheat—D. breviaristatum germplasm displayed reduced plant height, increased tillers and superior grain protein and gluten contents, improved gluten performance index. The results showed considerable potential for utilization of D. breviaristatum chromosome 1Vb segments in future wheat improvement.

The unique 75 K γ-secalins encoded by Sec2 loci in Secale species is composed of almost half rye storage proteins. The chromosomal location of Sec2 loci in wild Secale species, Secale africanum, was carried out by the wheat—S. africanum derivatives, which were identified by genomic in situ hybridization and multi-color fluorescence in situ hybridization. The Sec2 gene-specific PCR analysis indicated that the S. cereale Sec2 was located on chromosome 2R, while the S. africanum Sec2 was localized on chromosome 6Rafr of S. africanum. A total of 38 Sec2 gene sequences were isolated from S. africanum, S. cereale and S. sylvestre by PCR-based cloning. Phylogenetic analysis showed that S. africanum Sec2 diverged from S. cereale Sec2 approximately 2–3 million years ago. The illegitimate recombination of chromosome 2R–6R involving the Sec2 loci region may accelerate sequence variation during evolutionary process from wild to cultivated Secale species.

A new wheat-Thinopyrum translocation line CH13-21 was selected from the progenies derived from a cross between wheat-Th. intermedium partial amphiploid TAI7047 and wheat line Mianyang11. CH13-21 was characterized by using genomic in situ hybridization (GISH), multicolor-GISH (mc-GISH), multicolor-fluorescence in situ hybridization (mc-FISH) and chromosome-specific molecular markers. When inoculated with stripe rust and powdery mildew isolates, CH13-21 displayed novel resistance to powdery mildew and stripe rust which inherited from its Thinopyrum parent. The chromosomal counting analyses indicated that CH13-21 has 42 chromosomes, with normal bivalent pairing at metaphase I of meiosis. GISH probed by Th. intermedium genomic DNA showed that CH13-21 contained a pair of wheat-Th. intermedium translocated chromosomes. Sequential mc-FISH analyses probed by pSc119.2 and pAs1 clearly revealed that chromosome arm 6BS of CH13-21 was replaced by Thinopyrum chromatin in the translocation chromosome. The molecular markers analysis further confirmed that the introduced Th. intermedium chromatin in CH13-21 belonged to the long arm of homoeologous group 6 chromosome. Therefore, CH13-21 was a new T6BS.6Ai#1L compensating Robertsonian translocation line. It concludes that CH13-21 is a new genetic resource for wheat breeding programs providing novel variation for disease resistances.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating fungal diseases of wheat worldwide. It is essential to discover more sources of stripe rust resistance genes for wheat breeding programs. Specific locus amplified fragment sequencing (SLAF-seq) is a powerful tool for the construction of high-density genetic maps. In this study, a set of 200 recombinant inbred lines (RILs) derived from a cross between wheat cultivars Chuanmai 42 (CH42) and Chuanmai 55 (CH55) was used to construct a high-density genetic map and to identify quantitative trait loci (QTLs) for stripe rust resistance using SLAF-seq technology. A genetic map of 2828.51 cM, including 21 linkage groups, contained 6732 single nucleotide polymorphism markers (SNP). Resistance QTLs were identified on chromosomes 1B, 2A, and 7B; Qyr.saas-7B was derived from CH42, whereas Qyr.saas-1B and Qyr.saas-2A were from CH55. The physical location of Qyr.saas-1B, which explained 6.24−34.22% of the phenotypic variation, overlapped with the resistance gene Yr29. Qyr.saas-7B accounted for up to 20.64% of the phenotypic variation. Qyr.saas-2A, a minor QTL, was found to be a likely new stripe rust resistance locus. A significant additive effect was observed when all three QTLs were combined. The combined resistance genes could be of value in breeding wheat for stripe rust resistance.

To determine the composition of chromosome aberrations in a wheat‒Dasypyrum breviaristatum substitution line with seeds treated by a dose of gamma-rays (200 Gy), sequential non-denaturing fluorescence in situ hybridization (ND-FISH) with multiple oligonucleotide probes was used to screen individual plants of the mutagenized progenies. We identified 122 types of chromosome rearrangements, including centromeric, telomeric, and intercalary chromosome translocations from a total of 772 M1 and 872 M2 plants. The frequency of reciprocal translocations between B- and D-chromosomes was higher than that between A- and D-chromosomes. Eight translocations between D. breviaristatum and wheat chromosomes were also detected. The 13 stable plants with multiple chromosome translocations displayed novel agronomic traits. The newly developed materials will enhance wheat breeding programs through wheat‒Dasypyrum introgression and also facilitate future studies on the genetic and epigenetic effects of translocations in wheat genomics.

Thinopyrum has been widely used to improve wheat (Triticum aestivum L.) cultivars. Non-denaturing fluorescence in situ hybridization (ND-FISH) technology using oligonucleotides (oligo) as probes provides a convenient and efficient way to identify alien chromosomes in wheat backgrounds. However, suitable ND-FISH-positive oligo probes for distinguishing Thinopyrum chromosomes from wheat are lacking. Two oligo probes, Oligo-B11 and Oligo-pThp3.93, were designed according to the published Thinopyrum ponticum (Th. ponticum)-specific repetitive sequences. Both Oligo-B11 and Oligo-pThp3.93 can be used for ND-FISH analysis and can replace conventional GISH and FISH to discriminate some chromosomes of Th. elongatum, Th. intermedium, and Th. ponticum in wheat backgrounds. The two oligo probes provide a convenient way for the utilization of Thinopyrum germplasms in future wheat breeding programs.

The wild species, Thinopyrum intermedium. (Genome StStJSJSJJ), serves as a valuable germplasm resource providing novel genes for wheat improvement. In the current study, non-denaturing fluorescence in situ hybridization (ND-FISH) with multiple probes and comparative molecular markers were applied to characterize two wheat-Th. intermedium chromosome additions. Sequential ND-FISH with new labeled Th. intermedium specific oligo-probes were used to precisely determine the chromosomal constitution of Th. intermedium, wheat—Th. intermedium partial amphiploids and addition lines Hy36 and Hy37. The ND-FISH results showed that the added JS-St translocated chromosomes in Hy36 had minor Oligo-5S ribosomal DNA (rDNA) signals at the short arm, while a pair of J-St chromosomes in Hy37 had major Oligo-pTa71 and minor Oligo-5S rDNA signals. The 90K SNP array and PCR-based molecular markers that mapped on wheat linkage group 5 and 3 facilitated the identification of Thinopyrum chromosome introgressions in the addition lines, and confirmed that added chromosomes in Hy36 and Hy37 were 5JSS.3StS and 5JS.3StS, respectively. Complete coding sequences at the paralogous puroindoline-a (Pina) loci from Th. intermedium were cloned and localized on the short arm of chromosome 5JS of Hy36. Line Hy36 showed a reduction in the hardness index, which suggested that Th. intermedium-specific Pina gene sequences may be associated with the softness trait in wheat background. The molecular cytogenetic identification of novel wheat—Th. intermedium derivatives indicated that the frequent chromosome rearrangement occurred in the progenies of wheat-Thinopyrum hybridization. The new wheat-Thinopyrum derived lines may increase the genetic diversity for wheat breeding.

A large proportion of the genomes of grasses is comprised of tandem repeats (TRs), which include satellite DNA. A mini-satellite DNA sequence with a length of 44 bp, named Ta-3A1, was found to be highly accumulated in wheat genome, as revealed by a comprehensive sequence analysis. The physical distribution of Ta-3A1 in chromosomes 3A, 5A, 5B, 5D, and 7A of wheat was confirmed by nondenaturing fluorescence in situ hybridization (ND-FISH) after labeling the oligonucleotide probe. The analysis of monomer variants indicated that rapid sequence amplification of Ta-3A1 occurred first on chromosomes of linkage group 5, then groups 3 and 7. Comparative ND-FISH analysis suggested that rapid changes occurred in copy number and chromosomal locations of Ta-3A1 among the different species in the tribe Triticeae, which may have been associated with chromosomal rearrangements during speciation and polyploidization. The labeling and subsequent use of Ta-3A1 by ND-FISH may assist in the precise identification and documentation of novel wheat germplasm engineered by chromosome manipulation.

Wild barley eibi1 mutant with HvABCG31 gene mutation has low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. To better understand how such a mutant plant survives, we performed a genome-wide gene expression analysis. The leaf transcriptomes between the near-isogenic lines eibi1 and the wild type were compared using the 22-k Barley1 Affymetrix microarray. We found that the pleiotropic effect of the single gene HvABCG31 mutation was linked to the co-regulation of metabolic processes and stress-related system. The cuticle development involved cytochrome P450 family members and fatty acid metabolism pathways were significantly up-regulated by the HvABCG31 mutation, which might be anticipated to reduce the levels of cutin monomers or wax and display conspicuous cuticle defects. The candidate genes for responses to stress were induced by eibi1 mutant through activating the jasmonate pathway. The down-regulation of co-expressed enzyme genes responsible for DNA methylation and histone deacetylation also suggested that HvABCG31 mutation may affect the epigenetic regulation for barley development. Comparison of transcriptomic profiling of barley under biotic and abiotic stresses revealed that the functions of HvABCG31 gene to high-water loss rate might be different from other osmotic stresses of gene mutations in barley. The transcriptional profiling of the HvABCG31 mutation provided candidate genes for further investigation of the physiological and developmental changes caused by the mutant.

The hybrid between Brassica napus and B. rapa displays obvious heterosis in both growth performance and stress tolerances. A comparative transcriptome analysis for B. napus (AnAnCC genome), B. rapa (ArAr genome), and its hybrid F1 (AnArC genome) was carried out to reveal the possible molecular mechanisms of heterosis at the gene expression level. A total of 40,320 nonredundant unigenes were identified using B. rapa (AA genome) and B. oleracea (CC genome) as reference genomes. A total of 6,816 differentially expressed genes (DEGs) were mapped in the A and C genomes with 4,946 DEGs displayed nonadditively by comparing the gene expression patterns among the three samples. The coexistence of nonadditive DEGs including high-parent dominance, low-parent dominance, overdominance, and underdominance was observed in the gene action modes of F1 hybrid, which were potentially related to the heterosis. The coexistence of multiple gene actions in the hybrid was observed and provided a list of candidate genes and pathways for heterosis. The expression bias of transposable element-associated genes was also observed in the hybrid compared to their parents. The present study could be helpful for the better understanding of the determination and regulation of mechanisms of heterosis to aid Brassica improvement.

Powdery mildew (PM) is a very destructive disease of wheat (Triticum aestivum L.). Wheat-Thinopyrum ponticum introgression line CH7086 was shown to possess powdery mildew resistance possibly originating from Th. ponticum. Genomic in situ hybridization and molecular characterization of the alien introgression failed to identify alien chromatin. To study the genetics of resistance, CH7086 was crossed with susceptible genotypes. Segregation in F2 populations and F2:3 lines tested with Chinese Bgt race E09 under controlled conditions indicated that CH7086 carries a single dominant gene for powdery mildew resistance. Fourteen SSR and EST-PCR markers linked with the locus were identified. The genetic distances between the locus and the two flanking markers were 1.5 and 3.2 cM, respectively. Based on the locations of the markers by nullisomic-tetrasomic and deletion lines of 'Chinese Spring', the resistance gene was located in deletion bin 2BL-0.89-1.00. Conserved orthologous marker analysis indicated that the genomic region flanking the resistance gene has a high level of collinearity to that of rice chromosome 4 and Brachypodium chromosome 5. Both resistance specificities and tests of allelism suggested the resistance gene in CH7086 was different from previously reported powdery mildew resistance genes on 2BL, and the gene was provisionally designated PmCH86. Molecular analysis of PmCH86 compared with other genes for resistance to Bgt in the 2BL-0.89-1.00 region suggested that PmCH86 may be a new PM resistance gene, and it was therefore designated as Pm51. The closely linked flanking markers could be useful in exploiting this putative wheat-Thinopyrum translocation line for rapid transfer of Pm51 to wheat breeding programs.

The objective of this research was to characterize the high level of resistance to stripe that has been observed in the released wheat cultivar, Chuanmai45. A combination of classic genetic analysis, molecular and cytogenetic methods were used to characterize resistance in an F2 population derived from Chuanmai45 and the susceptible Chuanmai42. Inheritance of resistance was shown to be conferred by two genes in Chuanmai45. Fluorescence in situ hybridization (FISH) was used along with segregation studies to show that one gene was located on a 1RS.1BL translocation. Molecular markers were employed to show that the other locus was located on chromosome 4B. The defeated gene, Yr24/26, on chromosome 1BL was present in the susceptible parent and lines that recombined this gene with the 1RS.1BL translocation were identified. The germplasm, loci, and associated markers identified in this study will be useful for application in breeding programs utilizing marker-assisted selection.