Investigation of chromosomal genetic characteristics and identification of structural variation in the offspring of hexaploid triticale×hexaploid wheat.

Hexaploid triticale is an important genetic resource for genetic improvement of common wheat, which can broaden the genetic basis of wheat. In order to lay a foundation for the subsequent research and utilization of triticale germplasm materials, the chromosomal genetic characteristics of cross and backcross offspring of hexaploid triticale×hexaploid wheat were investigated in the process of transferring rye chromatin from hexaploid triticale to hexaploid wheat. Hybrid and backcross combinations were prepared with hexaploid triticale 16yin171 as the maternal parent and hexaploid wheat Chuanmai62 as the paternal parent. The chromosomes in root tip cells of F1, BC1F1 and BC1F2 plants were traced and identified non-denaturing florescence in situ hybridization (ND-FISH). The results indicated that the backcross setting rate of hybrid F1 was 2.61%. The transmission frequency of 2R chromosome was the highest in BC1F1 plants while the transmissibility of rye chromosome in BC1F2 plant was 6R>4R>2R, and the 5B-7B wheat translocation in BC1F2 plants showed severe segregation. A total of 24 structural variant chromosomes were observed both in BC1F1 and BC1F2 plants, including chromosome fragments, isochromosomes, translocations, and dicentric chromosomes. In addition, the seed length and 1000-grain weight of some BC1F2 plants were better than that of the hexaploid wheat parent Chuanmai 62. Therefore, multiple backcrosses should be adopted as far as possible to make the rapid recovery of group D chromosomes, ensuring the recovery of fertility in offspring, when hexaploid tritriale is used as a bridge to introduce rye genetic material into common wheat. At the same time, the potential application value of chromosomal structural variation materials should be also concerned.

A major yellow rust resistance QTL on chromosome 6A shows increased frequency in recent Norwegian spring wheat cultivars and breeding lines.

KEY MESSAGE: A major yellow rust resistance QTL, QYr.nmbu.6A, contributed consistent adult plant resistance in field trials across Europe, China, Kenya and Mexico. Puccinia striiformis f. sp. tritici, causing wheat yellow rust (YR), is one of the most devastating biotrophic pathogens affecting global wheat yields. Owing to the recent epidemic of the PstS10 race group in Europe, yellow rust has become a reoccurring disease in Norway since 2014. As all stage resistances (ASR) (or seedling resistances) are usually easily overcome by pathogen evolution, deployment of durable adult plant resistance (APR) is crucial for yellow rust resistance breeding. In this study, we assessed a Nordic spring wheat association mapping panel (n = 301) for yellow rust field resistance in seventeen field trials from 2015 to 2021, including nine locations in six countries across four different continents. Nine consistent QTL were identified across continents by genome-wide association studies (GWAS). One robust QTL on the long arm of chromosome 6A, QYr.nmbu.6A, was consistently detected in nine out of the seventeen trials. Haplotype analysis of QYr.nmbu.6A confirmed significant QTL effects in all tested environments and the effect was also validated using an independent panel of new Norwegian breeding lines. Increased frequency of the resistant haplotype was found in new varieties and breeding lines in comparison to older varieties and landraces, implying that the resistance might have been selected for due to the recent changes in the yellow rust pathogen population in Europe.

An efficient Oligo-FISH painting system for revealing chromosome rearrangements and polyploidization in Triticeae.

A chromosome-specific painting technique has been developed which combines the most recent approaches of the companion disciplines of molecular cytogenetics and genome research. We developed seven oligonucleotide (oligo) pools derivd from single-copy sequences on chromosomes 1 to 7 of barley (Hordeum vulgare L.) and corresponding collinear regions of wheat (Triticum aestivum L.). The seven groups of pooled oligos comprised between 10 986 and 12 496 45-bp monomers, and these then produced stable fluorescence in situ hybridization (FISH) signals on chromosomes of each linkage group of wheat and barley. The pooled oligo probes were applied to high-throughput karyotyping of the chromosomes of other Triticeae species in the genera Secale, Aegilops, Thinopyrum, and Dasypyrum, and the study also extended to some wheat-alien amphiploids and derived lines. We demonstrated that a complete set of whole-chromosome oligo painting probes facilitated the study of inter-species chromosome homologous relationships and visualized non-homologous chromosomal rearrangements in Triticeae species and some wheat-alien species derivatives. When combined with other non-denaturing FISH procedures using tandem-repeat oligos, the newly developed oligo painting techniques provide an efficient tool for the study of chromosome structure, organization, and evolution among any wild Triticeae species with non-sequenced genomes.

Genome-wide association mapping of leaf rust and stripe rust resistance in wheat accessions using the 90K SNP array.

KEY MESSAGE: A genome-wide association analysis identified diverse loci for seedling and adult plant resistance to leaf rust and stripe rust. KASP markers were developed and validated for marker-assisted selection. Wheat leaf rust and stripe rust cause significant losses in many wheat producing regions worldwide. The objective of this study was to identify chromosome regions conferring resistance to both leaf rust and stripe rust at the seedling and adult plant stages. A diversity panel of 268 wheat lines, including 207 accessions from different wheat growing regions in China, and 61 accessions from foreign countries, were evaluated for leaf rust response at seedling stage using eight Chinese Puccinia triticina pathotypes, and also tested for leaf rust and stripe rust at adult plant stage in multiple field environments. The panel was genotyped with the Wheat 90 K Illumina iSelect SNP array. Genome-wide association mapping (GWAS) was performed using the mixed linear model (MLM). Twenty-two resistance loci including the known Lr genes, Lr1, Lr26, Lr3ka, LrZH22, and 18 potentially new loci were identified associated with seedling resistance, explaining 4.6 to 25.2% of the phenotypic variance. Twenty-two and 23 adult plant resistance (APR) QTL associated with leaf and stripe rust, respectively, were identified at adult stage, explaining 4.2-11.5% and 4.4-9.7% of the phenotypic variance. Among them, QLr-2BS was the potentially most valuable all-stage resistance gene. Seven and six consistent APR QTL were identified in multiple environments including best linear unbiased prediction (BLUP) data, respectively. Comparison with previously mapped resistance loci indicated that three of the seven leaf rust resistance APR QTL, and two of the six stripe rust resistance APR QTL were new. Four potentially pleiotropic APR QTL, including Lr46/Yr29, QLr-2AL.1/QYr-2AL.1, QLr-2AL.2/QYr-2AL.2, and QLr-5BL/QYr-5BL.1, were identified. Twelve associated SNPs were converted into kompetitive allele-specific PCR (KASP) markers and verified in bi-parental populations. The study reports genetic loci conferring resistance to both diseases, and the closely linked markers should be applicable for marker-assisted wheat breeding.

Molecular dissection of Secale africanum chromosome 6Rafr in wheat enabled localization of genes for resistance to powdery mildew and stripe rust.

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.

Physical location of tandem repeats in the wheat genome and application for chromosome identification.

MAIN CONCLUSION: A general distribution of tandem repeats (TRs) in the wheat genome was predicted and a new web page combined with fluorescence in situ hybridization experiments, and the newly developed Oligo probes will improve the resolution for wheat chromosome identification. Comprehensive sequence analysis of tandem repeats (TR) in the wheat reference genome permits discovery and application of TRs for chromosome identification. Genome-wide localization of TRs was identified in the reference sequences of Chinese Spring using Tandem Repeat Finder (TRF). A database of repeats unit size, array number, and physical coverage length of TRs in the wheat genome was built. The distribution of TRs occupied 3-5% of the wheat chromosomes, with non-random dispersal across the A, B, and D genomes. Three classes of TRs surrounding the predicted genes were compared. An optimized computer-assisted website page B2DSC was constructed for the general distribution and chromosomally enriched zones of TR sequences to be displayed graphically. The physical distribution of predicted TRs in the wheat genome by B2DSC matched well with the corresponding hybridization signals obtained with fluorescence in situ hybridization (FISH). We developed 20 oligonucleotide probes representing 20-60 bp lengths of high copy number of TRs and verified by FISH. An integrated physical map of TR-Oligo probes for wheat chromosome identification was constructed. Our results suggest that the combination of both molecular cytogenetics and genomic research will significantly benefit wheat breeding through chromosome manipulation and engineering.

Identification of QTLs for Stripe Rust Resistance in a Recombinant Inbred Line Population.

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.

Precise identification of wheat - Thinopyrum intermedium translocation chromosomes carrying resistance to wheat stripe rust in line Z4 and its derived progenies.

The wheat - Thinopyrum intermedium derived line Z4 has displayed novel and effective stripe rust resistance for over 40 years. This study aimed to precisely identify the chromosome constitution of line Z4 and determine the stripe rust resistance contribution using multicolor fluorescent in situ hybridization (FISH) and molecular marker analysis. The results indicated that the Z4 line (2n = 44) contained two pairs of non-Robertsonian translocations without the 3A chromosomes of wheat. FISH karyotypes of F3 progenies derived from crosses between Z4 and MY11 indicated that the transmission of the translocated chromosomes appeared normal and the number of wheat chromosomes 3A and 3D frequently varied. The FISH signal distribution of a new repetitive probe, named Oligo-3A1, confirmed the physical breakage points on chromosome 3AL incorporated into translocated chromosomes. PLUG markers revealed the breakage points on chromosomes 3A, 7JS, and 3D invloved in the translocated chromosomes, and they were designated as T3DS-3AS.3AL-7JSS and T3AL-7JSS.7JSL. Stripe rust resistances surveys indicated that the proximal region of 7JSS or 7JSL may confer the resistance at the adult plant stage. The precise characterization of the chromosome complements of wheat - Th. intermedium Z4 and derived progenies has demonstrated the importance of combining cytogenetic and molecular approaches in the genomics era for further wheat genetic manipulation and breeding purposes.

Introduction of Thinopyrum intermedium ssp. trichophorum chromosomes to wheat by trigeneric hybridization involving Triticum, Secale and Thinopyrum genera.

MAIN CONCLUSION: Fluorescence in situ hybridization and molecular markers have confirmed that several chromosomes from Thinopyrum intermedium ssp. trichophorum have been added to a wheat background, which originated from a cross between a wheat- Thinopyrum partial amphiploid and triticale. The lines displayed blue grains and resistance to wheat stripe rust. Thinopyrum intermedium has been used as a valuable resource for improving the disease resistance and yield potential of wheat. With the aim to transfer novel genetic variation from Th. intermedium species for sustainable wheat breeding, a new trigeneric hybrid was produced by crossing an octoploid wheat-Th. intermedium ssp. trichophorum partial amphiploid with hexaploid triticale. Fluorescence in situ hybridization (FISH) revealed that Thinopyrum chromosomes were transmitted preferably and the number of rye chromosomes tended to decrease gradually in the selfed derivatives of the trigeneric hybrids. Four stable wheat-Th. intermedium chromosome substitution, addition and translocation lines were selected, and a 2JS addition line, two substitution lines of 4JS(4B) and 4J(4B), and a small 4J.4B translocation line were identified by FISH and molecular markers. It was revealed that the gene(s) responsible for blue grains may located on the FL0.60-1.00 of long arm of Th. intermedium-derived 4J chromosome. Disease resistance screenings indicated that chromosomes 4JS and 2JS appear to enhance the resistance to stripe rust in the adult plant stage. The new germplasm with Th. intermedium introgression shows promise for utilization of Thinopyrum chromosome segments in future wheat improvement.

New molecular markers and cytogenetic probes enable chromosome identification of wheat-Thinopyrum intermedium introgression lines for improving protein and gluten contents.

MAIN CONCLUSION: New molecular markers were developed for targeting Thinopyrum intermedium 1St#2 chromosome, and novel FISH probe representing the terminal repeats was produced for identification of Thinopyrum chromosomes. Thinopyrum intermedium has been used as a valuable resource for improving the disease resistance and yield potential of wheat. A wheat-Th. intermedium ssp. trichophorum chromosome 1St#2 substitution and translocation has displayed superior grain protein and wet gluten content. With the aim to develop a number of chromosome 1St#2 specific molecular and cytogenetic markers, a high throughput, low-cost specific-locus amplified fragment sequencing (SLAF-seq) technology was used to compare the sequences between a wheat-Thinopyrum 1St#2 (1D) substitution and the related species Pseudoroegneria spicata (St genome, 2n = 14). A total of 5142 polymorphic fragments were analyzed and 359 different SLAF markers for 1St#2 were predicted. Thirty-seven specific molecular markers were validated by PCR from 50 randomly selected SLAFs. Meanwhile, the distribution of transposable elements (TEs) at the family level between wheat and St genomes was compared using the SLAFs. A new oligo-nucleotide probe named Oligo-pSt122 from high SLAF reads was produced for fluorescence in situ hybridization (FISH), and was observed to hybridize to the terminal region of 1St#L and also onto the terminal heterochromatic region of Th. intermedium genomes. The genome-wide markers and repetitive based probe Oligo-pSt122 will be valuable for identifying Thinopyrum chromosome segments in wheat backgrounds.

Characterization of wheat-Secale africanum chromosome 5R(a) derivatives carrying Secale specific genes for grain hardness.

MAIN CONCLUSION: New wheat- Secale africanum chromosome 5R (a) substitution and translocation lines were developed and identified by fluorescence in situ hybridization and molecular markers, and chromosome 5R (a) specific genes responsible for grain hardness were isolated. The wild species, Secale africanum Stapf. (genome R(a)R(a)), serves as a valuable germplasm resource for increasing the diversity of cultivated rye (S. cereale L., genome RR) and providing novel genes for wheat improvement. In the current study, fluorescence in situ hybridization (FISH) and molecular markers were applied to characterize new wheat-S. africanum chromosome 5R(a) derivatives. Labeled rye genomic DNA (GISH) and the Oligo-probes pSc119.2 and pTa535 (FISH) were used to study a wheat-S. africanum amphiploid and a disomic 5R(a) (5D) substitution, and to identify a T5DL.5R(a)S translocation line and 5R(a)S and 5R(a)L isotelosome lines. Twenty-one molecular markers were mapped to chromosome 5R(a) arms which will facilitate future rapid identification of 5R(a) introgressions in wheat backgrounds. Comparative analysis of the molecular markers mapped on 5R(a) with homoeologous regions in wheat confirmed a deletion on the chromosome T5DL.5R(a)S, which suggests that the wheat-S. africanum Robertsonian translocation involving homologous group 5 may not be fully compensating. Complete coding sequences at the paralogous puroindoline-a (Pina) and grain softness protein gene (Gsp-1) loci from S. africanum were cloned and localized onto the short arm of chromosome 5R(a). The S. africanum chromosome 5R(a) substitution and translocation lines showed a reduction in the hardness index, which may be associated with the S. africanum- specific Pina and Gsp-1 gene sequences. The present study reports the production of novel wheat-S. africanum chromosome 5R(a) stripe rust resistant derivatives and new rye-specific molecular markers, which may find application in future use of wild Secale genome resources for grain quality studies and disease resistance breeding.

Characterization of Stripe Rust Resistance Genes in the Wheat Cultivar Chuanmai45.

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.

A novel wheat-Dasypyrum breviaristatum substitution line with stripe rust resistance.

The introduction of genetic variation from wild and cultivated Triticeae species has been a long-standing approach for wheat improvement. Dasypyrum breviaristatum species harbor novel and agronomically important genes for resistance against multi-fungal diseases. The development of new wheat-D. breviaristatum introgression lines offers chances for the identification of stripe rust resistance gene(s). A wheat line, D11-5, was selected from a cross between wheat line MY11 and wheat-D. breviaristatum partial amphiploid TDH-2. It was characterized by FISH and PCR-based molecular markers. Chromosome counting revealed that the D11-5 line shows a hexaploid set of 2n = 6x = 42 chromosomes. FISH analysis using the Dasypyrum repetitive sequence pDb12H as a probe demonstrated that D11-5 contained a pair of D. breviaristatum chromosomes, while FISH with wheat D-genomic repetitive sequences revealed that the chromosome 2D was absent in D11-5. The functional molecular markers confirmed that the introduced D. breviaristatum chromosomes belong to the homoeologous group 2, indicating that D11-5 was a 2V(b) (2D) disomic substitution line. Field resistance showed that the introduced D. breviaristatum chromosomes 2V(b) were responsible for the stripe rust resistance at the adult plant stage. FISH, C-banding, and PCR-based molecular marker analysis indicated that the chromosome 2V(b) of D. breviaristatum was completely different from the chromosome 2V of D. villosum. The identified wheat-D. breviaristatum chromosome substitution line D11-5 may be applied to produce agronomically desirable stripe rust resistance germplasm.

Comparative transcriptome analysis to reveal genes involved in wheat hybrid necrosis.

Wheat hybrid necrosis is an interesting genetic phenomenon that is found frequently and results in gradual death or loss of productivity of wheat. However, the molecular basis and mechanisms of this genetic phenomenon are still not well understood. In this study, the transcriptomes of wheat hybrid necrosis F1 and its parents (Neimai 8 and II469) were investigated using digital gene expression (DGE). A total of 1300 differentially expressed genes were identified, indicating that the response to hybrid necrosis in wheat is complicated. The assignments of the annotated genes based on Gene Ontology (GO) revealed that most of the up-regulated genes belong to "universal stress related", "DNA/RNA binding", "protein degradation" functional groups, while the down-regulated genes belong to "carbohydrate metabolism" and "translation regulation" functional groups. These findings suggest that these pathways were affected by hybrid necrosis. Our results provide preliminarily new insight into the underlying molecular mechanisms of hybrid necrosis and will help to identify important candidate genes involved in wheat hybrid necrosis.

Genome architecture of the allotetraploid wild grass Aegilops ventricosa reveals its evolutionary history and contributions to wheat improvement.

The allotetraploid wild grass Aegilops ventricosa (2n = 4x = 28, genome DvDvNvNv) has been recognized as an important germplasm resource for wheat improvement owing to its ability to tolerate biotic stresses. In particular, the 2NvS segment from Ae. ventricosa, as a stable and effective resistance source, has contributed greatly to wheat improvement. The 2NvS/2AS translocation is a prevalent chromosomal translocation between common wheat and wild relatives, ranking just behind the 1B/1R translocation in importance for modern wheat breeding. Here, we assembled a high-quality chromosome-level reference genome of Ae. ventricosa RM271 with a total length of 8.67 Gb. Phylogenomic analyses revealed that the progenitor of the Dv subgenome of Ae. ventricosa is Ae. tauschii ssp. tauschii (genome DD); by contrast, the progenitor of the D subgenome of bread wheat (Triticum aestivum L.) is Ae. tauschii ssp. strangulata (genome DD). The oldest polyploidization time of Ae. ventricosa occurred ∼0.7 mya. The Dv subgenome of Ae. ventricosa is less conserved than the D subgenome of bread wheat. Construction of a graph-based pangenome of 2AS/6NvL (originally known as 2NvS) segments from Ae. ventricosa and other genomes in the Triticeae enabled us to identify candidate resistance genes sourced from Ae. ventricosa. We identified 12 nonredundant introgressed segments from the Dv and Nv subgenomes using a large winter wheat collection representing the full diversity of the European wheat genetic pool, and 29.40% of European wheat varieties inherit at least one of these segments. The high-quality RM271 reference genome will provide a basis for cloning key genes, including the Yr17-Lr37-Sr38-Cre5 resistance gene cluster in Ae. ventricosa, and facilitate the full use of elite wild genetic resources to accelerate wheat improvement.

Development of a Set of Wheat-Rye Derivative Lines from Hexaploid Triticale with Complex Chromosomal Rearrangements to Improve Disease Resistance, Agronomic and Quality Traits of Wheat.

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.

Genetic Diversity Assessment of the International Maize and Wheat Improvement Center and Chinese Wheat Core Germplasms by Non-Denaturing Fluorescence In Situ Hybridization.

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.

Precise Identification of Chromosome Constitution and Rearrangements in Wheat-Thinopyrum intermedium Derivatives by ND-FISH and Oligo-FISH Painting.

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.

Molecular and Cytogenetic Identification of Wheat-Thinopyrum intermedium Double Substitution Line-Derived Progenies for Stripe Rust Resistance.

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.

Location of Tandem Repeats on Wheat Chromosome 5B and the Breakpoint on the 5BS Arm in Wheat Translocation T7BS.7BL-5BS Using Single-Copy FISH Analysis.

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.