Non-denaturing Fluorescence In Situ Hybridization Research Articles

Chromosomal analysis of progenies between Lilium intersectional hybrids and wild species using ND-FISH and GISH.

Intersectional hybrids in lilies possess significant breeding value, but the lack of complete lily genomes and complex genotypes pose challenges for early identification of lily hybrids. This study aimed to use intersectional hybrid cultivars as female parents and wild lilies as male parents to facilitate early identification of hybrid offsprings and enhance the efficiency and convenience of the process. We investigated the nature of cross combinations using Non-denaturing Fluorescence In Situ Hybridization (ND-FISH) and Genomic In Situ Hybridization (GISH) techniques. Three novel oligonucleotide probes-Oligo-pTa794, Oligo-pITS and Oligo-telo-were developed for lily chromosome research. Our results demonstrated successful hybridization between wild lilies and intersectional hybrid cultivars, producing a total of 130 hybrid progenies. The combination of ND-FISH and GISH analyses effectively revealed the genomic composition of the hybrid progeny and determined the parental origin of specific chromosomes. This research provides significant guidance for lily breeding practices and offers a valuable reference for the application of ND-FISH and GISH techniques in interspecific hybridization breeding and molecular cytogenetic research across various plant species. The methods developed enable more precise, efficient, and convenient identification of hybrid offsprings.

Characterization of novel wheat-Thinopyrum ponticum introgression lines derived from partial amphiploid AUS6770 for resistance to stripe rust

The wild decaploid species Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey is an important source of genes against biotic and abiotic stresses affecting wheat. The wheat–Th. ponticum partial amphiploid AUS6770 shows resistance to multiple diseases, including stripe rust, stem rust, and powdery mildew. Mitotic chromosomes of AUS6770 were characterized by non-denaturing-fluorescence in situ hybridization (ND-FISH), and the individual Th. ponticum chromosomes 1Ae to 7Ae were karyotypically distinguished by Oligo-FISH painting using bulked oligo pools based on wheat-barley collinear regions. A novel stripe rust resistant line A155, derived from AUS6770, was found to have 44 chromosomes, including a pair of 2Ae chromosomes and a pair of 6B-6Ae translocations. To detect plants with transfer of resistance genes from A155 to wheat chromosomes, 1770 plants were developed from F2–F5 progenies of A155 crossed with the susceptible wheat cultivar MY11 and characterized with ND-FISH using multiple probes. A high frequency of transmission of chromosome 2Ae was observed, and 31 types of 2Ae chromosomal aberrations were identified using ND-FISH. Ten chromosomal bins on the 2Ae chromosome were determined from the deletion and translocation lines based on genome-based PCR markers. In combination with the evaluation of disease resistance, the gene(s) for stripe rust resistance was located on the FL0.79–1.00 of 2AeS and covers the corresponding region of 0–58.26 Mb in the reference genome of Th. elongatum. The newly identified wheat-Th. ponticum 2Ae translocation lines can be exploited as potential germplasm in wheat breeding for stripe rust resistance

Characterization of New Wheat-Thinopyrum intermedium Derivative Lines with Superior Genes for Stripe Rust and Powdery Mildew Resistance.

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 basis of an elite wheat cultivar Guinong 29 with harmonious improvement between multiple diseases resistance and other comprehensive traits

Fungal diseases, such as powdery mildew and rusts, significantly affect the quality and yield of wheat. Pyramiding diverse types of resistance genes into cultivars represents the preferred strategy to combat these diseases. Moreover, achieving collaborative improvement between diseases resistance, abiotic stress, quality, and agronomic and yield traits is difficult in genetic breeding. In this study, the wheat cultivar, Guinong 29 (GN29), showed high resistance to powdery mildew and stripe rust at both seedling and adult plant stages, and was susceptible to leaf rust at the seedling stage but slow resistance at the adult-plant stage. Meanwhile, it has elite agronomic and yield traits, indicating promising coordination ability among multiple diseases resistance and other key breeding traits. To determine the genetic basis of these elite traits, GN29 was tested with 113 molecular markers for 98 genes associated with diseases resistance, stress tolerance, quality, and adaptability. The results indicated that two powdery mildew resistance (Pm) genes, Pm2 and Pm21, confirmed the outstanding resistance to powdery mildew through genetic analysis, marker detection, genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), and homology-based cloning; the stripe rust resistance (Yr) gene Yr26 and leaf rust resistance (Lr) genes Lr1 and Lr46 conferred the stripe rust and slow leaf rust resistance in GN29, respectively. Meanwhile, GN29 carries dwarfing genes Rht-B1b and Rht-D1a, vernalization genes vrn-A1, vrn-B1, vrn-D1, and vrn-B3, which were consistent with the phenotypic traits in dwarf characteristic and semi-winter property; carries genes Dreb1 and Ta-CRT for stress tolerance to drought, salinity, low temperature, and abscisic acid (ABA), suggesting that GN29 may also have elite stress-tolerance ability; and carries two low-molecular-weight glutenin subunit genes Glu-B3b and Glu-B3bef which contributed to high baking quality. This study not only elucidated the genetic basis of the elite traits in GN29 but also verified the capability for harmonious improvement in both multiple diseases resistance and other comprehensive traits, offering valuable information for breeding breakthrough-resistant cultivars.

Cytological and molecular characterization of wheat lines carrying leaf rust and stem rust resistance genes Lr24 and Sr24

Previous studies showed that Australian wheat cultivars Janz and Sunco carry leaf rust and stem rust resistance genes Lr24 and Sr24 derived from Thinopyrum ponticum chromosome arm 3AgL. However, the size of the alien segments carrying Lr24 and Sr24 in the lines were not determined. In this study, we used non-denaturing fluorescence in situ hybridization (ND-FISH), genomic in situ hybridization (GISH), and PCR-based landmark unique gene (PLUG) markers to visualize the alien segments in Janz and Sunco, and further compared them with the segments in US cultivars Agent and Amigo. The fraction length (FL) of the alien translocation in Agent was 0.70–1.00, whereas those in Janz, Sunco, and Amigo were smaller, at FL 0.85–1.00. It was deduced that the alien gene RAg encoding for red grain color and rust resistance genes Lr24 and Sr24 on chromosome arm 3AgL were in bins of FL 0.70–0.85 and 0.85–1.00, respectively. We retrieved and extracted nucleotide-binding site-leucine-rich repeat (NBS-LRR) receptor genes corresponding to the region of Lr24 and Sr24 on chromosomes 3E, and 3J, 3Js and 3St from the reference genome sequences of Th. elongatum and Th. intermedium, respectively. A set of molecular markers developed for Lr24 and Sr24 from those extracted NBS-LRR genes will provide valuable information for fine mapping and cloning of these genes.

Molecular Cytogenetic Characterization of Novel Wheat-Rye T1RS.1AL Translocation Lines with Resistance to Powdery Mildew and Stripe Rust Derived from the Chinese Rye Landrace Qinling.

Stripe rust and powdery mildew are serious diseases that severely decrease the yield of wheat. Planting wheat cultivars with powdery mildew and stripe rust resistance genes is the most effective way to control these two diseases. Introducing disease resistance genes from related species into the wheat genome via chromosome translocation is an important way to improve wheat disease resistance. In this study, nine novel T1RS.1AL translocation lines were developed from the cross of wheat cultivar Chuannong25 (CN25) and a Chinese rye Qinling. The results of non-denaturing fluorescence in situ hybridization and PCR showed that all new lines were homozygous for the T1RS.1AL translocation. These new T1RS.1AL translocation lines exhibited strong resistance to stripe rust and powdery mildew. The cytogenetics results indicated that the resistance of the new lines was conferred by the 1RS chromosome arms, which came from Qinling rye. The genetic analysis indicated that there were new dominant resistance genes on the 1RS chromosome arm resistant to stripe rust and powdery mildew, and their resistance patterns were different from those of Yr9, Pm8, and Pm17 genes. In addition, the T1RS.1AL translocation lines generally exhibited better agronomic traits in the field relative to CN25. These T1RS.1AL translocations have great potential in wheat-breeding programs in the future.

Identification and transfer of resistance to Fusarium head blight from Elymus repens chromosome arm 7StL into wheat

Fusarium head blight (FHB), mainly caused by Fusarium graminearum (Fg), is one of the most devastating fungal diseases in wheat production worldwide. Elymus repens (2n=6x=42, StStStStHH) is a wild relative of wheat with many biotic and abiotic stress resistance traits. To transfer and apply the wild germplasm's resistance gene (s) for wheat breeding, we identified a new translocation line K140-7 with high resistance to FHB, developed from the derivative progenies of E. repens crossed with common wheat cultivars. Cytogenetic analyses based on genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), oligonucleotide-FISH painting (Oligo-FISH painting), and single-gene FISH revealed that K140-7 had 40 wheat chromosomes and two 7DS·7StL translocated chromosomes. Wheat 55K SNP array analysis confirmed that the translocated breakpoint (340.8~342.5 Mb) was close to the centromere region of chromosome 7D (336.3~341.7 Mb), supporting the 7DS·7StL translocation event. Based on the diploid reference St genome of Pseudoroegneria libanotica, we developed 21 simple sequence repeats (SSR) markers, specific for chromosome arm 7StL. Genotyping and phenotyping analysis of the 7DS·7StL translocation in different wheat backgrounds demonstrated that the chromosome arm 7StL confers FHB resistance and possesses the dominant FHB resistance locus (s) named QFhb.Er-7StL. We further transferred QFhb.Er-7StL into three different wheat cultivars, their second 7DS·7StL translocation line-generations showed improved agronomic traits, representing new germplasms that could be used in wheat FHB-resistant breeding programs.

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.

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.

Molecular and cytogenetic dissection of stripe rust resistance gene Yr83 from rye 6R and generation of resistant germplasm in wheat breeding.

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.

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.

Development and Molecular Cytogenetic Characterization of a Novel Wheat-Rye T6RS.6AL Translocation Line from Secale cereale L. Qinling with Resistance to Stripe Rust and Powdery Mildew.

In this study, a novel T6RS.6AL translocation line, 117-6, was selected from a cross between common Chuannong25 (CN25) wheat and Qinling rye. The results of nondenaturing fluorescence in situ hybridization (ND-FISH) and PCR showed that 117-6 contained two T6RS.6AL translocation chromosomes. The distal region of the 6RS chromosome in 117-6 was mutant and showed different FISH signal patterns. When inoculated with different stripe rust races and powdery mildew races in seedlings, 117-6 expressed high resistance to them. The 117-6 line also exhibited high resistance to stripe rust and powdery mildew in the field under natural Puccinia striiformis f. sp. tritici (Pst) and Blumeria graminis f. sp. tritici (Bgt) infection. The cytogenetic analysis indicated that the introduction of 6RS conferred resistance ability. Compared with wheat parent CN25, 117-6 exhibited excellent agronomic traits in the field. The present study indicated that Qinling rye may carry favorite genes as a potential source for wheat genetic improvement, and 117-6 could be a useful germplasm for wheat breeding programs in the future.

Molecular cytogenetic characterization of a new wheat-Thinopyrum intermedium homoeologous group-6 chromosome disomic substitution line with resistance to leaf rust and stripe rust.

Thinopyrum intermedium (JJJsJsStSt, 2n = 6x = 42), a member of tertiary gene pool of hexaploid wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), provides several beneficial genes for wheat improvement. In this study, line CH51 was developed from the BC1F8 progeny of a partial wheat-Th. intermedium amphiploid TAI8335 (2n = 56) and wheat cultivar (cv.) Jintai 170. Somatic metaphase chromosome counting showed that CH51 had stable 42 chromosomes. Genomic in situ hybridization (GISH) analysis showed that CH51 had 40 wheat chromosomes and two Th. intermedium chromosomes involving translocation between Js- and St-genome chromosomes. Non-denaturing fluorescence in situ hybridization (ND-FISH) analysis revealed that CH51 lacked a pair of wheat chromosome 6B. Wheat 55K SNP array analysis verified that chromosome 6B had the highest percentage of missing SNP loci in both CH51 and Chinese Spring (CS) nullisomic 6B-tetrasomic 6D (CS-N6BT6D) and had the highest percentage of polymorphic SNP loci between CH51 and cv. Jintai 170. We identified that CH51 was a wheat-Th. intermedium T6StS.6JsL (6B) disomic substitution line. Disease resistance assessment showed that CH51 exhibited high levels of resistance to the prevalent Chinese leaf rust and stripe rust races in the field. Therefore, the newly developed line CH51 can be utilized as a potential germplasm in wheat disease resistance breeding.

Variations of wheat (Triticum aestivum L.) chromosomes caused by the 5A chromosomes with complex cytological structure.

To study the effects of structural alterations of chromosomes caused by tandem repeats on the meiotic recombination, the wheat (Triticum aestivum L.) 5A chromosomes with different structure from ten wheat cultivars were used to investigate their meiotic recombination using non-denaturing fluorescence in situ hybridization (ND-FISH) technology. Fifteen cross combinations were carried out and they were divided into seven F1 categories. The structural difference between the intercalary regions of the long arms of the two 5A chromosomes (5AL) in the F1 categories III, VI, and VII was greater than that in the categories I and II, subsequently, the recombination frequencies in the distal regions of the 5AL arm in the progenies from the three categories were significantly lower than that from the categories I and II. For the two 5A chromosomes in the F1 categories VI and VII, the structural differences in the distal regions of both of the two arms were greater than that in the categories IV and V. So, the recombination frequencies in the intercalary region of the 5AL arm in the progeny from the categories IV and V were higher than that in the progeny from the categories VI and VII. The breakage of 5A chromosome together with the 5A translocations and the breakage of some other chromosomes were observed in the progeny from the F1 categories V, VI, and VII. These chromosomal variations were not observed in the progenies from the other four F1 categories. In conclusion, the smaller structural difference between the 5A chromosomes in distal regions of the two arms resulted in a higher recombination frequency in interstitial region and vice versa. The 5A chromosome with complex cytological structure can be used to induce genetic variations of wheat genome.

Molecular cytogenetic identification of new wheat-rye 6R, 6RS, and 6RL addition lines with resistance to stripe rust and powdery mildew.

Stripe rust and powdery mildew are devastating diseases that have severe effects on wheat production. Introducing resistant genes/loci from wheat-related species into the wheat genome is an important method to improve wheat resistance. Rye (Secale cereale L.) is a cross-pollinating plant and is the most important related species for wheat genetic improvement. In this study, we developed three 6RS ditelosomic addition lines, three 6RL ditelosomic addition lines, and two 6R disomic addition lines by crossing common wheat cultivar Chuannong 25 and rye inbred line QL2. The chromosome composition of all new lines was confirmed by non-denaturing fluorescence in situ hybridization (ND-FISH) and molecular marker analyses. Disease responses to different Puccinia striiformis f. sp. tritici (Pst) races and Blumeria graminis f. sp. tritici (Bgt) isolates and cytogenetic analysis showed that the resistance of the new lines was derived from the rye chromosome 6R of QL2, and both arms (6RS and 6RL) may harbor resistance genes against Pst and Bgt. These new lines could be used as a promising bridging parent and valuable genetic resource for wheat disease resistance improvement.

Development and application of universal ND-FISH probes for detecting P-genome chromosomes based on Agropyron cristatum transposable elements.

Fluorescence in situ hybridization (FISH) is a basic tool that is widely used in cytogenetic research. The detection efficiency of conventional FISH is limited due to its time-consuming nature. Oligonucleotide (oligo) probes with fluorescent labels have been applied in non-denaturing FISH (ND-FISH) assays, which greatly streamline experimental processes and save costs and time. Agropyron cristatum, which contains one basic genome, "P," is a vital wild relative for wheat improvement. However, oligo probes for detecting P-genome chromosomes based on ND-FISH assays have not been reported. In this study, according to the distribution of transposable elements (TEs) in Triticeae genomes, 94 oligo probes were designed based on three types of A. cristatum sequences. ND-FISH validation showed that 12 single oligo probes generated a stable and obvious hybridization signal on whole P chromosomes in the wheat background. To improve signal intensity, mixed probes (Oligo-pAc) were prepared by using the 12 successful probes and validated in the diploid accession A. cristatum Z1842, a small segmental translocation line and six allopolyploid wild relatives containing the P genome. The signals of Oligo-pAc covered the entire chromosomes of A. cristatum and were more intense than those of single probes. The results indicate that Oligo-pAc can replace conventional genomic in situ hybridization (GISH) probes to identify P chromosomes or segments in non-P-genome backgrounds. Finally, we provide a rapid and efficient method specifically for detecting P chromosomes in wheat backgrounds by combining the Oligo-pAc probe with the Oligo-pSc119.2-1 and Oligo-pTa535-1 probes, which can replace conventional sequential GISH/FISH assays. Altogether, we developed a set of oligo probes based on the ND-FISH assays to identify P-genome chromosomes, which can promote utilization of A. cristatum in wheat improvement programs.

A Novel Wheat-Rye 2R (2D) Disomic Substitution Line Pyramids Two Types of Resistance to Powdery Mildew.

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a devastating disease of wheat that seriously affects yield and quality worldwide. Because of the extensive growth of wheat cultivars with homogeneous genetic background, exploring novel resistant resources from wheat relatives has become important for increasing the genetic diversity of wheat. Rye (Secale cereale) is a wheat relative possessing abundant resistance genes because of its high variation. Wheat line AL69, resistant to powdery mildew, was developed by crossing, backcrossing, and self-pollination for multiple generations between hexaploid triticale Zhongsi 237 and common wheat cultivar Zimai 17. Through genomic in situ hybridization (GISH) and multicolor fluorescence in situ hybridization (FISH), nondenaturing FISH, multicolor GISH, and selection with specific molecular markers, AL69 was determined to be a wheat-rye 2R (2D) disomic substitution line. Testing with different B. graminis f. sp. tritici isolates and genetic analysis showed that the all-stage resistance (also called seedling resistance) of AL69 was conferred by the cataloged powdery mildew resistance gene Pm4b derived from Zimai 17, and its adult-plant resistance was derived from the alien chromosome 2R of Zhongsi 237, which was found to be different from the previously reported rye-derived Pm genes, including Pm7 on 2RL. In addition, AL69 showed improved spike number per plant, spike length, fertile spikelet number per spike, kernel number per spike, and grain yield per plant compared with its wheat parent Zimai 17. An elite line S251 combining powdery mildew resistance with excellent agronomic performance was selected from the progenies of AL69 and wheat cultivar Jimai 22. Therefore, AL69 has two types of resistance genes to powdery mildew and improved agronomic traits through pyramiding and thus can be used as a promising genetic stock for wheat breeding.

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.

Molecular Cytogenetic Identification of the Wheat–Dasypyrum villosum T3DL·3V#3S Translocation Line with Resistance against Stripe Rust

The annual species Dasypyrum villosum possesses several potentially valuable genes for the improvement of common wheat. Previously, we identified a new stripe rust-resistant line, the Chinese Spring (CS)–D. villosum 3V#3 (3D) substitution line (named CD-3), and mapped its potential rust resistance gene (designated as YrCD-3) on the 3V#3 chromosome originating from D. villosum. The objective of the present study was to further narrow down the YrCD-3 locus to a physical region and develop wheat-3V#3 introgression lines with strong stripe rust resistance. By treating CD-3 seeds with 60Co γ-irradiation, two CS-3V#3 translocation lines, T3V#3S.3DL and T3DS.3V#3L (termed 22-12 and 24-20, respectively), were identified from the M4 generation through a combination of non-denaturing fluorescence in situ hybridization (ND-FISH) and functional molecular markers. Stripe rust resistance tests showed that the line 22-12 exhibited strong stripe rust resistance similarly to CD-3, whereas 24-20 was susceptible to stripe rust similarly to CS, indicating that YrCD-3 is located on the short arm of 3V#3. The line 22-12 can potentially be used for further wheat improvement. Additionally, to trace 3V#3 in the wheat genetic background, we produced 30 3V#3-specific sequence tag (EST) markers, among which, 11 markers could identify 3V#3S. These markers could be valuable in fine-mapping YrCD-3.

Origins and chromosome differentiation of Thinopyrum elongatum revealed by PepC and Pgk1 genes and ND-FISH.

Thinopyrum elongatum is an important gene pool for wheat genetic improvement. However, the origins of the Thinopyrum genomes and the nature of the genus' intraspecific relationships are still controversial. In this study, we used single-copy nuclear genes and non-denaturing fluorescence in situ hybridization (ND-FISH) to characterize genome constitution and chromosome differentiation in Th.elongatum. According to phylogenetic analyses based on PepC and Pgk1 genes, there was an E genome with three versions (Ee, Eb, Ex) and St genomes in the polyploid Th.elongatum. The ND-FISH results of pSc119.2 and pAs1 revealed that the karyotypes of diploid Th.elongatum and Th.bessarabicum were different, and the chromosome differentiation occurred among accessions of the diploid Th.elongatum. In addition, the tetraploid Th.elongatum has two groups of ND-FISH karyotype, indicating that the tetraploid Th.elongatum might be a segmental allotetraploid. In summary, our results suggested that the diploid Th.elongatum, Th.Bessarabicum, and Pseudoroegneria were the donors of the Ee, Eb, and St genomes to the polyploid Th.elongatum, respectively.