Disomic Substitution Lines Research Articles

Development and identification of a novel wheat-Thinopyrum ponticum disomic substitution line DS5Ag(5D) with new genes conferring resistance to powdery mildew and leaf rust

BackgroundPowdery mildew (caused by Blumeria graminis f. sp. tritici (Bgt)) and leaf rust (caused by Puccinia triticina (Pt)) are prevalent diseases in wheat (Triticum aestivum L.) production. Thinopyrum ponticum (2n = 10x = 70, EeEeEbEbExExStStStSt) contains genes that confer high levels of resistance to these diseases.ResultsAn elite wheat-Th. ponticum disomic substitution line, DS5Ag(5D), was developed in the Bainong Aikang 58 (AK58) background. The line was assessed using genomic in situ hybridization (GISH), oligo-nucleotide probe multiplex (ONPM) fluorescence in situ hybridization (FISH), and molecular markers. Twenty eight chromosome-specific molecular markers were identified for the alien chromosome, and 22 of them were co-dominant. Additionally, SNP markers from the wheat 660 K SNP chip were utilized to confirm chromosome identification and they provide molecular tools for tagging the chromosome in concern. The substitution line demonstrated high levels of resistance to powdery mildew throughout its growth period and to leaf rust at the adult stage. Based on the resistance evaluation of five F5 populations between the substitution lines and wheat genotypes with different levels of sensitivity to the two diseases. Results showed that the resistance genes located on 5Ag confered stable resistance against both diseases across different backgrounds. Resistance spectrum analysis combined with diagnostic marker detection of known resistance genes of Th. ponticum revealed that 5Ag contained two novel genes, Pm5Ag and Lr5Ag, which conferred resistance to powdery mildew and leaf rust, respectively.ConclusionsIn this study, a novel wheat-Th. ponticum disomic substitution line DS5Ag(5D) was successfully developed. The Th. ponticum chromosome 5Ag contain new resistance genes for powdery mildew and leaf rust. Chromosomic—specific molecular markers were generated and they can be used to track the 5Ag chromosome fragments. Consequently, this study provides new elite germplasm resources and molecular markers to facilitate the breeding of wheat varieties that is resistant to powdery mildew and leaf rust.

Development and characterisation of novel durum wheat-H. chilense 4Hch chromosome lines as a source for resistance to Septoria tritici blotch.

The use of wild species as a source of genetic variability is a valued tool in the framework of crop breeding. Hordeum chilense Roem. et Schult is a wild barley species that can be a useful genetic donor for sustainable wheat breeding which carries genes conferring resistance to some diseases or increasing grain quality, among others. Septoria tritici blotch (STB), caused by the Zymoseptoria tritici fungus, is one of the most important wheat diseases worldwide, affecting both bread and durum wheat and having a high economic impact. Resistance to STB has been previously described in H. chilense chromosome 4Hch. In this study, we have developed introgression lines for H. chilense chromosome 4Hch in durum wheat using interspecific crosses, advanced backcrosses, and consecutive selfing strategies. Alien H. chilense chromosome segments have been reduced in size by genetic crosses between H. chilense disomic substitution lines in durum wheat and durum wheat lines carrying the Ph1 deletion. Hordeum chilense genetic introgressions were identified in the wheat background through several plant generations by fluorescence in situ hybridisation (FISH) and simple sequence repeat (SSR) markers. An STB infection analysis has also been developed to assess STB resistance to a specific H. chilense chromosome region. The development of these H. chilense introgression lines with moderate to high resistance to STB represents an important advance in the framework of durum breeding and can be a valuable tool for plant breeders.

Introgression of an All-Stage and Broad-Spectrum Powdery Mildew Resistance Gene Pm3VS from Dasypyrum villosum Chromosome 3V into Wheat.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a serious disease that threatens wheat production globally. It is imperative to explore novel resistance genes to control this disease by developing and planting resistant varieties. Here, we identified a wheat-Dasypyrum villosum 3V (3D) disomic substitution line, NAU3815 (2n = 42), with a high level of powdery mildew resistance at both the seedling and adult-plant stages. Subsequently, NAU3815 was used to generate recombination between chromosomes 3V and 3D. Through genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), and 3VS- and 3VL-specific markers analysis, four introgression lines were developed from the selfing progenies of 3V and 3D double monosomic line NAU3816, which was derived from the F1 hybrids of NAU3815/NAU0686. There were t3VS (3D) ditelosomic substitution line NAU3817, t3VL (3D) ditelosomic substitution line NAU3818, homozygous T3DL·3VS translocation line NAU3819, and homozygous T3DS·3VL translocation line NAU3820. Powdery mildew tests of these lines confirmed the presence of an all-stage and broad-spectrum powdery mildew resistance gene, Pm3VS, located on chromosome arm 3VS. When compared with the recurrent parent NAU0686 plants, the T3DL·3VS translocation line NAU3819 showed no obvious negative effect on yield-related traits. However, the introduction of the T3DL·3VS translocated chromosome had a strong effect on reducing the flag-leaf length. Consequently, the T3DL·3VS translocation line NAU3819 provides a new germplasm in breeding for both resistance and plant architecture.

Introgression of an adult-plant powdery mildew resistance gene Pm4VL from Dasypyrum villosum chromosome 4V into bread wheat.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) seriously threatens wheat production worldwide. It is imperative to identify novel resistance genes from wheat and its wild relatives to control this disease by host resistance. Dasypyrum villosum (2n = 2x = 14, VV) is a relative of wheat and harbors novel genes for resistance against multi-fungal diseases. In the present study, we developed a complete set of new wheat-D. villosum disomic introgression lines through genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and molecular markers analysis, including four disomic substitution lines (2n=42) containing respectively chromosomes 1V#6, 2V#6, 3V#6, and 6V#6, and four disomic addition lines (2n=44) containing respectively chromosomes 4V#6, 5V#6, 6V#6 and 7V#6. These lines were subsequently evaluated for their responses to a mixture Bgt isolates at both seedling and adult-plant stages. Results showed that introgression lines containing chromosomes 3V#6, 5V#6, and 6V#6 exhibited resistance at both seedling and adult-plant stages, whereas the chromosome 4V#6 disomic addition line NAU4V#6-1 exhibited a high level of adult plant resistance to powdery mildew. Moreover, two translocation lines were further developed from the progenies of NAU4V#6-1 and the Ph1b mutation line NAU0686-ph1b. They were T4DL·4V#6S whole-arm translocation line NAU4V#6-2 and T7DL·7DS-4V#6L small-fragment translocation line NAU4V#6-3. Powdery mildew tests of the two lines confirmed the presence of an adult-plant powdery mildew resistance gene, Pm4VL, located on the terminal segment of chromosome arm 4V#6L (FL 0.6-1.00). In comparison with the recurrent parent NAU0686 plants, the T7DL·7DS-4V#6L translocation line NAU4V#6-3 showed no obvious negative effect on yield-related traits, providing a new germplasm in breeding for resistance.

Registration of WGC002 spring wheat containing wild grass‐derived Fusarium head blight resistance gene Fhb7The2

AbstractThe USDA‐ARS and North Dakota State University Agricultural Experiment Station jointly released the Fusarium head blight (FHB)‐resistant spring wheat (Triticum aestivum L.) germplasm WGC002 (Reg. no. GP‐1089, PI 702949) in May 2023. WGC002 is a wheat‐Thinopyrum elongatum 7B‐7E translocation line, designated 7BS·7BL‐7EL, with the wheat chromosome 7BL terminal region replaced by the homoeologous counterpart of the Th. elongatum chromosome 7EL that contains the novel FHB resistance gene Fhb7The2. WGC002 was developed from the Chinese Spring (CS) wheat‐Th. elongatum disomic substitution line DS 7E(7B) using our genomics‐enabled chromosome engineering pipeline. The pedigree of WGC002 is DS 7E(7B)/2*CS ph1b mutant//DS 7E(7B). WGC002 has consistently exhibited resistance to FHB in inoculations of greenhouse grown plants. WGC002 does not contain the yellow flour pigment gene in the Fhb7The2 haplotype present on the terminal 7EL segment of the 7BS·7BL‐7EL translocation and has not exhibited any obvious linkage drag on the 7EL segment. Therefore, WGC002 is ready for immediate use in wheat breeding. One sequence‐tagged site (STS) and two polymerase chain reaction allelic competitive extension markers were developed specifically for Fhb7The2 and validated in different wheat genotypes. They are highly diagnostic for Fhb7The2 and extremely useful in marker‐assisted introgression of Fhb7The2 in wheat breeding. In summary, WGC002 is a new wild grass‐derived FHB‐resistant spring wheat germplasm with diagnostic DNA markers available to conduct marker‐assisted selection of Fhb7The2, that will enhance and diversify FHB resistance of wheat.

Development and characterization of a line with substitution of chromosome 4B of wheat Triticum aestivum L. on chromosome 4Hmar of wild barley Hordeum marinum ssp. gussoneanum (4x).

Introgressive hybridization is the main method of broadening the genetic diversity of bread wheat. Wild barley Hordeum marinum ssp. gussoneanum Hudson (2n = 4x = 28) has useful agronomical traits, such as high resistance to stress factors, that could be a potential source of new genes for bread wheat improvement. This study aimed to evaluate the possibility of introgression of H. marinum chromosomes into the genome of bread wheat using an incomplete amphiploid H. marinum ssp. gussoneanum (4x)-T. aestivum (Pyrotrix 28) (2n = 54) carrying the cytoplasm of wild barley. For this purpose, we crossed the line of bread wheat variety Pyrotrix 28 with an incomplete amphiploid, and then selected cytogenetically stable 42-chromosome plants with a high level of fertility in hybrid progeny. Genomic in situ hybridization (GISH) revealed a pair of H. marinum chromosomes in the genome of these plants. C- banding analysis confirmed that bread wheat chromosome 4B was replaced by wild barley chromosome 4Hmar. SSR markers Xgwm368 and Xgwm6 confirmed the absence of chromosome 4B, and EST markers BAWU808 and BAW112 identified chromosome 4Hmar in the genome of the isolated disomic wheat-barley substitution line. The study of this line showed that the substitution of chromosome 4B with chromosome 4Hmar resulted in a change of some morphological traits. It included intense anthocyanin coleoptile coloration, specific for H. marinum, as well as a lack of purple coloration of the ears in the leaf sheath, specific for Pyrotrix 28. Line 4Hmar(4B) showed increased performance for several traits, including plant height, number of spikes and tillers per plant, spikelet and grain number in the main spike, grain number per plant, but it had decreased values of 1000-grain weight compared to wheat. Cytogenetic stability and fertility of line 4Hmar(4B) indicated a high compensation ability of barley 4Hmar for wheat chromosome 4B and confirmed their homeology.

Development and Characterization of a Novel Wheat-Tetraploid Thinopyrum elongatum 6E (6D) Disomic Substitution Line with Stripe Rust Resistance at the Adult Stage.

Stripe rust, which is caused by Puccinia striiformis f. sp. tritici, is one of the most devastating foliar diseases of common wheat worldwide. Breeding new wheat varieties with durable resistance is the most effective way of controlling the disease. Tetraploid Thinopyrum elongatum (2n = 4x = 28, EEEE) carries a variety of genes conferring resistance to multiple diseases, including stripe rust, Fusarium head blight, and powdery mildew, which makes it a valuable tertiary genetic resource for enhancing wheat cultivar improvement. Here, a novel wheat-tetraploid Th. elongatum 6E (6D) disomic substitution line (K17-1065-4) was characterized using genomic in situ hybridization and fluorescence in situ hybridization chromosome painting analyses. The evaluation of disease responses revealed that K17-1065-4 is highly resistant to stripe rust at the adult stage. By analyzing the whole-genome sequence of diploid Th. elongatum, we detected 3382 specific SSR sequences on chromosome 6E. Sixty SSR markers were developed, and thirty-three of them can accurately trace chromosome 6E of tetraploid Th. elongatum, which were linked to the disease resistance gene(s) in the wheat genetic background. The molecular marker analysis indicated that 10 markers may be used to distinguish Th. elongatum from other wheat-related species. Thus, K17-1065-4 carrying the stripe rust resistance gene(s) is a novel germplasm useful for breeding disease-resistant wheat cultivars. The molecular markers developed in this study may facilitate the mapping of the stripe rust resistance gene on chromosome 6E of tetraploid Th. elongatum.

Identification of a Small Translocation from 6R Possessing Stripe Rust Resistance to Wheat.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici Eriks. & E. Henn, is the most devastating fungal disease of bread wheat. Here, a wheat-rye multiple disomic substitution line, SLU126 4R (4D), 5R (5D), and 6R (7D), possessing resistance against 25 races of P. striiformis f. sp. tritici, was used and crossed with Chinese Spring ph1b to induce homeologous recombination to produce introgressions with a reduced rye chromosome segment. Seedling assays confirmed that the stripe rust resistance from SLU126 was retained over multiple generations. Through genotyping-by-sequencing (GBS) platforms and aligning the putative GBS-single-nucleotide polymorphism (SNPs) to the full-length annotated rye nucleotide-binding leucine-rich repeat (NLR) genes in the parental lines (CS ph1b, SLU126, CSA, and SLU820), we identified the physical position of 26, 13, and 9 NLR genes on chromosomes 6R, 4R, and 5R, respectively. The physical positions of 25 NLR genes on chromosome 6R were identified from 568,460,437 bp to 879,958,268 bp in the 6RL chromosome segment. Based on these NLR positions on the 6RL chromosome segment, the three linked SNPs (868,123,650 to 873,285,112 bp) were validated through kompetitive allele-specific PCR (KASP) assays in SLU126 and resistance plants in the family 29-N3-5. Using these KASP markers, we identified a small piece of the rye translocation (i.e., as a possible 6DS.6DL.6RL.6DL) containing the stripe resistance gene, temporary designated YrSLU, within the 6RL segment. This new stripe rust resistance gene provides an additional asset for wheat improvement to mitigate yield losses caused by stripe rust.

Physical mapping of a new powdery mildew resistance locus from Thinopyrum ponticum chromosome 4AgS.

Thinopyrum ponticum (Podp.) Barkworth and D.R. Dewey is a decaploid species that has served as an important genetic resource for improving wheat for the better part of a century. The wheat-Th. ponticum 4Ag (4D) disomic substitution line Blue 58, which was obtained following the distant hybridization between Th. ponticum and common wheat, has been stably resistant to powdery mildew under field conditions for more than 40 years. The transfer of 4Ag into the susceptible wheat cultivar Xiaoyan 81 resulted in powdery mildew resistance, indicating the alien chromosome includes the resistance locus. Irradiated Blue 58 pollen were used for the pollination of the recurrent parent Xiaoyan 81, which led to the development of four stable wheat-Th. ponticum 4Ag translocation lines with diverse alien chromosomal segments. The assessment of powdery mildew resistance showed that translocation line L1 was susceptible, but the other three translocation lines (WTT139, WTT146, and WTT323) were highly resistant. The alignment of 81 specific-locus amplified fragments to the Th. elongatum genome revealed that 4Ag originated from a group 4 chromosome. The corresponding physical positions of every 4Ag-derived fragment were determined according to a cytogenetic analysis, the amplification of specific markers, and a sequence alignment. Considering the results of the evaluation of disease resistance, the Pm locus was mapped to the 3.79-97.12 Mb region of the short arm of chromosome 4Ag. Because of its durability, this newly identified Pm locus from a group 4 chromosome of Th. ponticum may be important for breeding wheat varieties with broad-spectrum disease resistance.

Cytogenetic characterization and molecular marker development for a wheat-T. boeoticum 4Ab (4B) disomic substitution line with stripe rust resistance.

Triticum boeoticum (2n = 2x = 14, AbAb) is an important relative of wheat. This species tolerates many different types of environmental stresses, including drought, salt, and pathogenic infection, and is lower in dietary fiber and higher in antioxidants, protein (15-18%), lipids, and trace elements than common wheat. However, the gene transfer rate from this species to common wheat is low, and few species-specific molecular markers are available. In this study, the wheat-T. boeoticum substitution line Z1889, derived from a cross between the common wheat cultivar Crocus and Triticum boeoticum line G52, was identified using multicolor fluorescence in situ hybridization (mc-FISH), multicolor genomic in situ hybridization (mc-GISH) and a 55K single nucleotide polymorphism (SNP) array. Z1889 was revealed to be a 4Ab (4B) substitution line with a high degree of resistance to stripe rust pathogen strains prevalent in China. In addition, 22 4Ab chromosome-specific molecular markers and 11 T. boeoticum genome-specific molecular markers were developed from 1145 4Ab chromosome-specific fragments by comparing the sequences generated by specific-length amplified fragment sequencing (SLAF-seq), with an efficiency of up to 55.0%. Furthermore, the specificity of these markers was verified in four species containing the Ab genome. These markers can not only be used for the detection of the 4Ab chromosome but also provide a basis for molecular marker-assisted selection-based breeding in wheat.

Genetic analysis of resistance to powdery mildew on 7Mg chromosome of wheat–Aegilops geniculata, development and utilization of specific molecular markers

BackgroundPowdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is prevalent in the main wheat-producing regions of China, resulting in severe yield losses in recent years. Mining and utilization of resistant genes from wild relatives of wheat is the most environmentally sound measure to control disease. Aegilops geniculata Roth (2n = 2x = 28, UgUgMgMg) is an essential and valuable disease-resistance gene donor for wheat improvement as a close relative species.ResultsIn this study, to validate powdery mildew resistance locus on chromosome 7Mg, two genetic populations were constructed and through crossing wheat – Ae. geniculata 7Mg disomic addition line NA0973-5-4-1-2-9-1 and 7Mg (7 A) alien disomic substitution line W16998 with susceptible Yuanfeng175 (YF175, authorized varieties from Shaanxi province in 2005), respectively. Cytological examination, in situ hybridization (ISH), and functional molecular markers analysis revealed that the plants carrying chromosome 7Mg showed high resistance to powdery mildew in both F1 and F2 generation at the seedling stage. Besides, 84 specific markers were developed to identify the plants carrying chromosome 7Mg resistance based on the specific-locus amplified fragment sequencing (SLAF-seq) technique. Among them, four markers were selected randomly to check the reliability in F2 segregating populations derived from YF175/NA0973-5-4-1-2-9-1 and YF175/W16998. In summary, the above analysis confirmed that a dominant high powdery mildew resistance gene was located on chromosome 7Mg of Ae. geniculata.ConclusionThe results provide a basis for mapping the powdery mildew resistance gene mapping on chromosome 7Mg and specific markers for their utilization in the future.

Cytogenetic identification and molecular marker development of a novel wheat-Leymus mollis 4Ns(4D) alien disomic substitution line with resistance to stripe rust and Fusarium head blight.

Leymus mollis (Trin.) Pilg. (2n = 4x = 28, NsNsXmXm) potentially harbours useful genes that might contribute to the improvement of wheat. We describe M862 as a novel wheat-L. mollis alien disomic substitution line from a cross between wheat cv. 7182 and octoploid Tritileymus M47. Cytological observations indicate that M862 has a chromosome constitution of 2n = 42 = 21II. Two 4D chromosomes of wheat substituted by two L. mollis Ns chromosomes were observed, using the GISH and ND-FISH analyses. Molecular marker, 55K SNP array and wheat-P. huashanica liquid array (GenoBaits®WheatplusPh) analyses further indicate that the alien chromosomes are L. mollis 4Ns. Therefore, it was deduced that M862 was a wheat-L. mollis 4Ns(4D) alien disomic substitution line. There were also changes in chromosomes 1A, 1D, 2B and 5A detected by ND-FISH analysis. Transcriptome sequencing showed that the structural variation of 1D, 1A and 5A may have smaller impact on gene expression than that for 2B. In addition, a total of 16 markers derived from Lm#4Ns were developed from transcriptome sequences, and these proved to be highly effective for tracking the introduced chromosome. M862 showed reduced height, larger grains (weight and width), and was highly resistance to CYR32 and CYR34 stripe rust races at the seedling stage and mixed stripe rust races (CYR32, CYR33 and CYR34) at the adult stage. It was also resistance to Fusarium head blight (FHB). This alien disomic substitution line M862 may be exploited as an important genetic material in the domestication of stipe rust and FHB resistance wheat varieties.

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.

Identification and Characterization of Wheat-Aegilops comosa 7M (7A) Disomic Substitution Lines with Stripe Rust and Powdery Mildew Resistance.

Aegilops comosa (MM, 2n = 2x = 14), an important diploid species from the wheat tertiary gene pool, contains many unique genes/traits of potential use for wheat breeding, such as disease resistance. In this study, three sister lines, NAL-32, NAL-33, and NAL-34, were identified from a wheat-A. comosa distant cross using fluorescence in situ hybridization, simple sequence repeat markers, and PCR-based unique gene markers combined with single nucleotide polymorphism (SNP) array analysis. Genetically, NAL-32 contained neither an alien nor translocation chromosome, whereas NAL-33 and NAL-34 had disomic 7M (7A) substitution chromosomes but differed in the absence or presence of the 1BL/1RS translocation chromosomes, respectively. The absence of 7A in NAL-33 and NAL-34 and the unusual 1B in the latter were verified by wheat 55K SNP arrays. The two 7M (7A) substitution lines had similar levels of resistance to stripe rust and powdery mildew, but better than that of NAL-32 and their common wheat parents, suggesting that the stripe rust and powdery mildew resistance of NAL-33 and NAL-34 were derived from the 7M of A.comosa. This research provides important bridge materials that can potentially be used for transferring stripe rust and powdery mildew resistance.

Fine mapping of powdery mildew and stripe rust resistance genes Pm5V/Yr5V transferred from Dasypyrum villosum into wheat without yield penalty.

The novel wheat powdery mildew and stripe rust resistance genes Pm5V/Yr5V are introgressed from Dasypyrum villosum and fine mapped to a narrowed region in 5VS, and their effects on yield-related traits were characterized. The powdery mildew and stripe rust seriously threaten wheat production worldwide. Dasypyrum villosum (2n = 2x = 14, VV), a relative of wheat, is a valuable resource of resistance genes for wheat improvement. Here, we describe a platform for rapid introgression of the resistance genes from D. villosum into the wheat D genome. A complete set of new wheat-D. villosum V (D) disomic substitution lines and 11 D/V Robertsonian translocation lines are developed and characterized by molecular cytogenetic method. A new T5DL·5V#5S line NAU1908 shows resistance to both powdery mildew and stripe rust, and the resistances associated with 5VS are confirmed to be conferred by seedling resistance gene Pm5V and adult-plant resistance gene Yr5V, respectively. We flow-sort chromosome arm 5VS and sequence it using the Illumina NovaSeq 6000 system that allows us to generate 5VS-specific markers for genetic mapping of Pm5V/Yr5V. Fine mapping shows that Pm5V and Yr5V are closely linked and the location is narrowed to an approximately 0.9Mb region referencing the sequence of Chinese Spring 5DS. In this region, a NLR gene in scaffold 24,874 of 5VS orthologous to TraesCS5D02G044300 is the most likely candidate gene for Pm5V. Soft- and hard-grained T5DL·5V#5S introgressions confer resistance to both powdery mildew and stripe rust in diverse wheat genetic backgrounds without yield penalty. Meanwhile, significant decrease in plant height and increase in yield were observed in NIL-5DL·5V#5S compared with that in NIL-5DL·5DS. These results indicate that Pm5V/Yr5V lines might have the potential value to facilitate wheat breeding for disease resistance.

Assessing the Potential of Using the Langdon 5D(5B) Substitution Line for the Introgression of Aegilops tauschii Into Durum Wheat.

Aegilops tauschii, the D-genome donor of hexaploid wheat, provides a source of genetic variation that could be used for tetraploid (durum) wheat improvement. In addition to the genes for wheat quality on the D-genome, which differentiate between bread and durum wheats in terms of end-use properties, genes coding for resistances to biotic and abiotic stresses are also present on the D-genome which would be useful in durum wheat. The introgression of Ae. tauschii into durum wheat, however, requires cytogenetic manipulation to induce homoeologous chromosome pairing to promote recombination. For this purpose, the introgression of Ae. tauschii into durum wheat was performed through a bridge cross of the wild species to the Langdon 5D(5B) disomic substitution line that lacks the Ph1 locus present on chromosome 5B, followed by a cross of the F1 to the durum wheat cultivar Om Rabi 5. Subsequent generations were self-fertilized, and these were screened for D-genome introgressions using (i) D-genome-specific Kompetitive Allele-Specific PCR (KASP) markers and (ii) KASP markers polymorphic between the 5D chromosomes of wheat, present in the Langdon 5D(5B) substitution line, and of Ae. tauschii. Homozygous introgression lines were confirmed using genomic and fluorescence in situ hybridization. The results showed that the use of the Langdon 5D(5B) disomic substitution line did not promote D-genome introgression across all linkage groups with only a limited success in the introgression of Ae. tauschii 5D segments into durum wheat.

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.

Wheat-Thinopyrum Substitution Lines Imprint Compensation Both From Recipients and Donors.

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.

Molecular cytogenetics and development of St-chromosome-specific molecular markers of novel stripe rust resistant wheat\u2013Thinopyrum intermedium and wheat\u2013Thinopyrum ponticum substitution lines

BackgroundOwing to their excellent resistance to abiotic and biotic stress, Thinopyrum intermedium (2n = 6x = 42, JJJsJsStSt) and Th. ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs. Disomic substitution lines (DSLs) carrying one pair of alien chromosomes are valuable bridge materials for transmission of novel genes, fluorescence in situ hybridization (FISH) karyotype construction and specific molecular marker development.ResultsSix wheat–Thinopyrum DSLs derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), were characterized by sequential FISH–genome in situ hybridization (GISH), multicolor GISH (mc-GISH), and an analysis of the wheat 15 K SNP array combined with molecular marker selection. ES-9 (DS2St (2A)) and ES-10 (DS3St (3D)) are wheat–Th. ponticum DSLs, while ES-23 (DS2St (2A)), ES-24 (DS3St (3D)), ES-25(DS2St (2B)), and ES-26 (DS2St (2D)) are wheat–Th. intermedium DSLs. ES-9, ES-23, ES-25 and ES-26 conferred high thousand-kernel weight and stripe rust resistance at adult stages, while ES-10 and ES-24 were highly resistant to stripe rust at all stages. Furthermore, cytological analysis showed that the alien chromosomes belonging to the same homoeologous group (2 or 3) derived from different donors carried the same FISH karyotype and could form a bivalent. Based on specific-locus amplified fragment sequencing (SLAF-seq), two 2St-chromosome-specific markers (PTH-005 and PTH-013) and two 3St-chromosome-specific markers (PTH-113 and PTH-135) were developed.ConclusionsThe six wheat–Thinopyrum DSLs conferring stripe rust resistance can be used as bridging parents for transmission of valuable resistance genes. The utility of PTH-113 and PTH-135 in a BC1F2 population showed that the newly developed markers could be useful tools for efficient identification of St chromosomes in a common wheat background.

Development and Identification of a Novel Wheat-Thinopyrum scirpeum 4E (4D) Chromosomal Substitution Line with Stripe Rust and Powdery Mildew Resistance.

Stripe rust caused by Puccinia striiformis f. sp. tritici and powdery mildew caused by Blumeria graminis f. sp. tritici are devastating diseases of wheat worldwide. Exploration of new disease-resistant genes from cultivated wheat and wild relatives are the most effective means of reducing the amounts of fungicides applied to combat these diseases. Thinopyrum scirpeum (2n = 4x = 28, EEEE) is an important promising reservoir of useful genes, including stripe rust and powdery mildew resistance, and may be useful for increasing wheat disease resistance. Here, we characterize a novel wheat-Th. scirpeum disomic substitution line, K16-730-3, and chromosome-specific markers were developed that can be used to trace the Th. scirpeum chromosome or chromosome segments transferred into wheat. Genomic in situ hybridization and fluorescence in situ hybridization analyses indicated that K16-730-3 is a new 4E (4D) chromosomal substitution line. Evaluation of seedling and adult disease responses revealed that K16-730-3 is resistant to stripe rust and powdery mildew. In addition, no obvious difference in grain yield was observed between K16-730-3 and its wheat parents. Genotyping-by-sequencing analyses indicated that 74 PCR-based markers can accurately trace chromosome 4E, which were linked to the disease resistance genes in the wheat background. Further marker validation analyses revealed that 13 specific markers can distinguish between the E-genome chromosomes of Th. scirpeum and the chromosomes of other wheat-related species. The new substitution line K16-730-3 carrying the stripe rust and powdery mildew resistance genes will be useful as novel germplasm in breeding for disease resistance. The markers developed in this study can be used in marker-assisted selection for increasing disease resistance in wheat.