Identification and introgression of a novel leaf rust resistance gene from Thinopyrum intermedium chromosome 7Js into wheat.

KEY MESSAGE: A novel leaf rust resistance locus located on a terminal segment (0-69.29 Mb) of Thinopyrum intermedium chromosome arm 7JsS has been introduced into wheat genome for disease resistance breeding. Xiaoyan 78829, a wheat-Thinopyrum intermedium partial amphiploid, exhibits excellent resistance to fungal diseases in wheat. To transfer its disease resistance to common wheat (Triticum aestivum), we previously developed a translocation line WTT26 using chromosome engineering. Disease evaluation showed that WTT26 was nearly immune to 14 common races of leaf rust pathogen (Puccinia triticina) and highly resistant to Ug99 race PTKST of stem rust pathogen (P. graminis f. sp. tritici) at the seedling stage. It also displayed high adult plant resistance to powdery mildew (caused by Blumeria graminis f. sp. tritici). Cytogenetic and molecular marker analysis revealed that WTT26 carried a T4BS·7JsS chromosome translocation. Once transferred into the susceptible wheat genetic background, chromosome 7JsS exhibited its resistance to leaf rust, indicating that the resistance locus was located on this alien chromosome. To enhance the usefulness of this locus in wheat breeding, we further developed several new translocation lines with small Th. intermedium segments using irradiation and developed 124 specific markers using specific-locus amplified fragment sequencing, which increased the marker density of chromosome 7JsS. Furthermore, a refined physical map of chromosome 7JsS was constructed with 74 specific markers, and six bins were thus arranged according to the co-occurrence of markers and alien chromosome segments. Combining data from specific marker amplification and resistance evaluation, we mapped a new leaf rust resistance locus in the 0-69.29 Mb region on chromosome 7JsS. The translocation lines carrying the new leaf rust resistance locus and its linked markers will contribute to wheat disease-resistance breeding.

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.

Establishment and identification of six wheat-Thinopyrum ponticum disomic addition lines derived from partial amphiploid Xiaoyan 7430.

KEY MESSAGE: Six wheat-Thinopyrum ponticum disomic addition lines derived from partial amphiploid Xiaoyan 7430 were identified using in situ hybridization and SNP microarray, the homoeologous group and stripe rust resistance of each alien chromosome were determined, and Th. ponticum chromosome-specific markers were developed. Xiaoyan 7430 is a significant partial amphiploid, which is used to set up a bridge for transferring valuable genes from Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey into common wheat. To accelerate the application of these useful genes in enriching the genetic variability of cultivated wheat by chromosome engineering, a complete set of derived addition lines has been created from Xiaoyan 7430. The chromosome composition of each line was characterized by the combination of genomic in situ hybridization and multicolor fluorescence in situ hybridization (mc-FISH), and the homoeology of each alien chromosome was determined by wheat SNP microarray analysis. Addition line WTA55 with alien group-6 chromosome was evaluated resistant to stripe rust isolates at both the seedling and grain-filling stages (Zadoks scale at z.11 and z.73). Diagnostic marker analysis proved that it could carry a novel stripe rust resistance gene derived from Th. ponticum. Furthermore, a FISH probe and 45 molecular markers specific for alien chromosomes were developed based on specific-locus amplified fragment sequencing (SLAF-seq). Of which 27 markers were separately located on single alien chromosome, and some of them could be used to identify the derived translocation lines. This set of addition lines as well as the molecular markers and the FISH probe will promote the introgression of abundant variation from Th. ponticum into wheat in wheat improvement programs.

Cytogenetic identification and molecular marker development of a novel wheat-Thinopyrum ponticum translocation line with powdery mildew resistance.

KEY MESSAGE: A new wheat-Thinopyrum ponticum translocation line with excellent powdery mildew resistance was produced, and alien-specific PCR markers and FISH probes were developed by SLAF-seq. Powdery mildew is one of the most threatening diseases in wheat production. Thinopyrum ponticum (Podp.) Barkworth and D. R. Dewey, as a wild relative, has been used for wheat genetic improvement for the better part of a century. In view of the good powdery mildew resistance of Th. ponticum, we have been working to transfer the resistance genes from Th. ponticum to wheat by creating translocation lines. In this study, a new wheat-Th. ponticum translocation line with excellent resistance and agronomic performance was developed and through seedling disease evaluation, gene postulation and diagnostic marker analysis proved to carry a novel Pm gene derived from Th. ponticum. Cytogenetic analysis revealed that a small alien segment was translocated to the terminal of chromosome 1D to form new translocation TTh-1DS·1DL chromosome. The translocation breakpoint was determined to lie in 21.5 Mb region of chromosome 1D by using Wheat660K SNP array analysis. Based on specific-locus amplified fragment sequencing (SLAF-seq) technology, eight molecular markers and one repetitive sequence probe were developed, which were specific for Th. ponticum. Fortunately, the probe could be used in distinguishing six alien chromosome pairs in partial amphiploid Xiaoyan 7430 by fluorescence in situ hybridization (FISH). Furthermore, a Thinopyrum-specific oligonucleotide probe was designed depending on the sequence information of the FISH probe. The novel translocation line could be used in wheat disease resistance breeding, and these specific markers and probes will enable wheat breeders to rapidly trace the alien genome with the novel Pm gene(s).

Screening of Salt-Tolerant Thinopyrum ponticum Under Two Coastal Region Salinity Stress Levels.

To accelerate the exploitation and use of marginal soils and develop salt-tolerant forage germplasm suitable for the coastal regions of China, seven lines of decaploid tall wheatgrass [Thinopyrum ponticum (Podp.) Barkworth and D. R. Dewey, 2n = 10x = 70] were transplanted under low (.3%) and high (.5%) salt conditions for a comprehensive analysis at the adult-plant stage. Differences were observed among these materials, especially in terms of grass yield, agronomic characteristics, and physiological and biochemical indices. Line C2 grew best with the highest shoot total fresh and dry weights under all conditions except for the milk-ripe stage in Dongying in 2019. The total membership value of C2 also reflected its excellent performance after transplanting. As superior germplasm, its relatively high antioxidant enzyme activities and chlorophyll a/b ratio suggested C2 may maintain normal metabolic and physiological functions under saline conditions. Furthermore, decaploid tall wheatgrass as a forage grass species has a high nutritive value beneficial for animal husbandry. Accordingly, line C2 may be used as excellent germplasm to develop salt-tolerant cultivars in the Circum-Bohai sea.

Mapping QTL for seedling morphological and physiological traits under normal and salt treatments in a RIL wheat population.

KEY MESSAGE: The genetic basis of 27 seedling traits under normal and salt treatments was fully analyzed in a RIL wheat population, and seven QTL intervals were validated in two other genetic populations. Soil salinity seriously constrains wheat (Triticum aestivum L.) production globally by influencing its growth and development. To explore the genetic basis of salt tolerance in wheat, a recombinant inbred line (RIL) population derived from a cross between high-yield wheat cultivar Zhongmai 175 (ZM175) and salt-tolerant cultivar Xiaoyan 60 (XY60) was used to map QTL for seedling traits under normal and salt treatments based on a high-density genetic linkage map. A total of 158 stable additive QTL for 27 morphological and physiological traits were identified and distributed on all wheat chromosomes except 3A and 4D. They explained 2.35-46.43% of the phenotypic variation with a LOD score range of 2.61-40.38. The alleles from XY60 increased corresponding traits for 100 QTL, while the alleles from ZM175 had positive effects for the other 58 QTL. Nearly half of the QTL (78/158) were mapped in nine QTL clusters on chromosomes 2A, 2B, 2D, 4B, 5A, 5B, 5D, and 7D (2), respectively. To prove the reliability and potentiality in molecular marker-assisted selection (MAS), seven QTL intervals were validated in two other genetic populations. Besides additive QTL, 94 pairs of loci were detected with significant epistatic effect and 20 QTL were found to interact with treatment. This study provides a full elucidation of the genetic basis of seedling traits (especially root system-related traits) associated with salt tolerance in wheat, and the developed kompetitive allele-specific PCR markers closely linked to stable QTL would supply strong supports to MAS in salt-tolerant wheat breeding.

Chromosomal composition analysis and molecular marker development for the novel Ug99-resistant wheat-Thinopyrum ponticum translocation line WTT34.

KEY MESSAGE: A novel Ug99-resistant wheat-Thinopyrum ponticum translocation line was produced, its chromosomal composition was analyzed and specific markers were developed. Stem rust caused by Puccinia graminis f. sp. tritici Eriks. & E. Henn (Pgt) has seriously threatened global wheat production since Ug99 race TTKSK was first detected in Uganda in 1998. Thinopyrum ponticum is near immune to Ug99 races and may be useful for enhancing wheat disease resistance. Therefore, developing new wheat-Th. ponticum translocation lines that are resistant to Ug99 is crucial. In this study, a novel wheat-Th. ponticum translocation line, WTT34, was produced. Seedling and field evaluation revealed that WTT34 is resistant to Ug99 race PTKST. The resistance was derived from the alien parent Th. ponticum. Screening WTT34 with markers linked to Sr24, Sr25, Sr26, Sr43, and SrB resulted in the amplification of different DNA fragments from Th. ponticum, implying WTT34 carries at least one novel stem rust resistance gene. Genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization (mc-FISH), and multi-color GISH (mc-GISH) analyses indicated that WTT34 carries a T5DS·5DL-Th translocation, which was consistent with wheat660K single-nucleotide polymorphism (SNP) array results. The SNP array also uncovered a deletion event in the terminal region of chromosome 1D. Additionally, the homeology between alien segments and the wheat chromosomes 2A and 5D was confirmed. Furthermore, 51 PCR-based markers derived from the alien segments of WTT34 were developed based on specific-locus amplified fragment sequencing (SLAF-seq). These markers may enable wheat breeders to rapidly trace Th. ponticum chromosomal segments carrying Ug99 resistance gene(s).

Cytogenetic identification and molecular marker development for the novel stripe rust-resistant wheat-Thinopyrum intermedium translocation line WTT11.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. Xiaoyan 78829, a partial amphidiploid developed by crossing common wheat with Thinopyrum intermedium, is immune to wheat stripe rust. To transfer the resistance gene of this excellent germplasm resource to wheat, the translocation line WTT11 was produced by pollen irradiation and assessed for immunity to stripe rust races CYR32, CYR33 and CYR34. A novel stripe rust-resistance locus derived from Th. intermedium was confirmed by linkage and diagnostic marker analyses. Molecular cytogenetic analyses revealed that WTT11 carries a TTh·2DL translocation. The breakpoint of 1B was located at 95.5 MB, and the alien segments were found to be homoeologous to wheat-group chromosomes 6 and 7 according to a wheat660K single-nucleotide polymorphism (SNP) array analysis. Ten previously developed PCR-based markers were confirmed to rapidly trace the alien segments of WTT11, and 20 kompetitive allele-specific PCR (KASP) markers were developed to enable genotyping of Th. intermedium and common wheat. Evaluation of agronomic traits in two consecutive crop seasons uncovered some favorable agronomic traits in WTT11, such as lower plant height and longer main panicles, that may be applicable to wheat improvement. As a novel genetic resource, the new resistance locus may be useful for wheat disease-resistance breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00060-3.

Mapping QTL for agronomic traits under two levels of salt stress in a new constructed RIL wheat population.

KEY MESSAGE: QTL for 15 agronomic traits under two levels of salt stress in dry salinity field were mapped in a new constructed RIL population utilizing a Wheat55K SNP array. Furthermore, eight QTL were validated in a collected natural population. Soil salinity is one of the major abiotic stresses causing serious impact on crop growth, development and yield. As one of the three most important crops in the world, bread wheat (Triticum aestivum L.) is severely affected by salinity, too. In this study, an F7 recombinant inbred line (RIL) population derived from a cross between high-yield wheat cultivar Zhongmai 175 and salt-tolerant cultivar Xiaoyan 60 was constructed. The adult stage performances of the RIL population and their parent lines under low and high levels of salt stress were evaluated for three consecutive growing seasons. Utilizing a Wheat55K SNP array, a high-density genetic linkage map spinning 3250.71 cM was constructed. QTL mapping showed that 90 stable QTL for 15 traits were detected, and they were distributed on all wheat chromosomes except 4D, 6B and 7D. These QTL individually explained 2.34-32.43% of the phenotypic variation with LOD values ranging from 2.68 to 47.15. It was found that four QTL clusters were located on chromosomes 2D, 3D, 4B and 6A, respectively. Notably, eight QTL from the QTL clusters were validated in a collected natural population. Among them, QPh-4B was deduced to be an allele of Rht-B1. In addition, three kompetitive allele-specific PCR (KASP) markers derived from SNPs were successfully designed for three QTL clusters. This study provides an important base for salt-tolerant QTL (gene) cloning in wheat, and the markers, especially the KASP markers, will be useful for marker-assisted selection in salt-tolerant wheat breeding.