Genomic characterization and gene bank curation of Aegilops: the wild relatives of wheat.

Genetic diversity found in crop wild relatives is critical to preserve and utilize for crop improvement to achieve sustainable food production amid climate change and increased demand. We genetically characterized a large collection of 1,041 Aegilops accessions distributed among 23 different species using more than 45K single nucleotide polymorphisms identified by genotyping-by-sequencing. The Wheat Genetics Resource Center (WGRC) Aegilops germplasm collection was curated through the identification of misclassified and redundant accessions. There were 49 misclassified and 28 sets of redundant accessions within the four diploid species. The curated germplasm sets now have improved utility for genetic studies and wheat improvement. We constructed a phylogenetic tree and principal component analysis cluster for all Aegilops species together, giving one of the most comprehensive views of Aegilops. The Sitopsis section and the U genome Aegilops clade were further scrutinized with in-depth population analysis. The genetic relatedness among the pair of Aegilops species provided strong evidence for the species evolution, speciation, and diversification. We inferred genome symbols for two species Ae. neglecta and Ae. columnaris based on the sequence read mapping and the presence of segregating loci on the pertinent genomes as well as genetic clustering. The high genetic diversity observed among Aegilops species indicated that the genus could play an even greater role in providing the critical need for untapped genetic diversity for future wheat breeding and improvement. To fully characterize these Aegilops species, there is an urgent need to generate reference assemblies for these wild wheats, especially for the polyploid Aegilops.

Extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass.

BACKGROUND: An Italian ryegrass population from Arkansas, USA developed glyphosate resistance due to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification. The plants in this population with approximately 70 EPSPS copies were used in the present study for the physical mapping of amplified copies of EPSPS gene to determine the possible mechanism of EPSPS gene amplification conferring glyphosate resistance in Italian ryegrass. RESULT: Fluorescence in situ hybridization (FISH) analysis of glyphosate resistant (GR) Italian ryegrass plants with approximately 70 EPSPS copies displayed EPSPS hybridization signals randomly on most of the metaphase chromosomes. Glyphosate susceptible (GS) Italian ryegrass plants with one EPSPS copy displayed single prominent EPSPS hybridization signal, which was co-localized with 5S rDNA locus along with few additional signals on the outside of chromosomes. Pulsed-field gel electrophoresis (PFGE) followed by DNA blot using EPSPS gene as a probe identified a prominent EPSPS hybridization around the 400 kb region in GR DNA samples, but not in GS DNA samples. CONCLUSION: We report the extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass. Physical mapping of amplified copies of EPSPS gene in Italian ryegrass by FISH gives us a clue that the amplified copies of EPSPS gene may be present in the extrachromosomal DNA elements. Further analysis by PFGE followed by DNA blotting revealed that the extrachromosomal DNA containing EPSPS is approximately 400 kb similar in size with that of eccDNA replicon in Amaranthus palmeri. © 2023 Society of Chemical Industry.

Extrachromosomal circular DNA-mediated spread of herbicide resistance in interspecific hybrids of pigweed.

Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNA-powered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F1 hybrids of Amaranthus sp. The glyphosate-resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate-susceptible Amaranthus tuberculatus and GR Amaranthus palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in Amaranthus spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution.

Flow karyotyping of wheat-Aegilops additions facilitate dissecting the genomes of Ae. biuncialis and Ae. geniculata into individual chromosomes.

Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.

A high-throughput skim-sequencing approach for genotyping, dosage estimation and identifying translocations.

The development of next-generation sequencing (NGS) enabled a shift from array-based genotyping to directly sequencing genomic libraries for high-throughput genotyping. Even though whole-genome sequencing was initially too costly for routine analysis in large populations such as breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to capitalize on whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage to genotype various breeding populations, a limitation comes in the time and cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on the amount of data required, and could be extended to 3,072 samples or more. Panels of doubled haploid wheat lines (Triticum aestivum, CDC Stanley x CDC Landmark), wheat-barley (T. aestivum x Hordeum vulgare) and wheat-wheatgrass (Triticum durum x Thinopyrum intermedium) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1 × down to 0.01 × per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

Origin and genetic analysis of stem rust resistance in wheat line Tr129.

Wheat line Tr129 is resistant to stem rust, caused by Puccinia graminis f. sp. tritici (Pgt). The resistance in Tr129 was reportedly derived from Aegilops triuncialis, but the origin and genetics of resistance have not been confirmed. Here, genomic in situ hybridization (GISH) showed that no Ae. triuncialis chromatin was present in Tr129. Genetic and phenotypic analysis was conducted on F2 and DH populations from the cross RL6071/Tr129. Seedlings were tested with six Pgt races and were genotyped using an Illumina iSelect 90 K SNP array and kompetitive allele specific PCR (KASP) markers. Mapping and phenotyping showed that Tr129 carried four stem rust resistance (Sr) genes on chromosome arms 2BL (Sr9b), 4AL (Sr7b), 6AS (Sr8a), and 6DS (SrTr129). SrTr129 co-segregated with markers for SrCad, however Tr129 has a unique haplotype suggesting the resistance could be new. Analysis of a RL6071/Peace population revealed that like SrTr129, SrCad is ineffective against three North American races. This new understanding of SrCad will guide its use in breeding. Tr129 and the DNA markers reported here are useful resources for improving stem rust resistance in cultivars.

Genetic characterization and curation of diploid A-genome wheat species.

A-genome diploid wheats represent the earliest domesticated and cultivated wheat species in the Fertile Crescent and include the donor of the wheat A sub-genome. The A-genome species encompass the cultivated einkorn (Triticum monococcum L. subsp. monococcum), wild einkorn (T. monococcum L. subsp. aegilopoides (Link) Thell.), and Triticum urartu. We evaluated the collection of 930 accessions in the Wheat Genetics Resource Center (WGRC) using genotyping by sequencing and identified 13,860 curated single-nucleotide polymorphisms. Genomic analysis detected misclassified and genetically identical (>99%) accessions, with most of the identical accessions originating from the same or nearby locations. About 56% (n = 520) of the WGRC A-genome species collections were genetically identical, supporting the need for genomic characterization for effective curation and maintenance of these collections. Population structure analysis confirmed the morphology-based classifications of the accessions and reflected the species geographic distributions. We also showed that T. urartu is the closest A-genome diploid to the A-subgenome in common wheat (Triticum aestivum L.) through phylogenetic analysis. Population analysis within the wild einkorn group showed three genetically distinct clusters, which corresponded with wild einkorn races α, ß, and γ described previously. The T. monococcum genome-wide FST scan identified candidate genomic regions harboring a domestication selection signature at the Non-brittle rachis 1 (Btr1) locus on the short arm of chromosome 3Am at ∼70 Mb. We established an A-genome core set (79 accessions) based on allelic diversity, geographical distribution, and available phenotypic data. The individual species core set maintained at least 79% of allelic variants in the A-genome collection and constituted a valuable genetic resource to improve wheat and domesticated einkorn in breeding programs.

Development of DNA Markers From Physically Mapped Loci in Aegilops comosa and Aegilops umbellulata Using Single-Gene FISH and Chromosome Sequences.

Breeding of agricultural crops adapted to climate change and resistant to diseases and pests is hindered by a limited gene pool because of domestication and thousands of years of human selection. One way to increase genetic variation is chromosome-mediated gene transfer from wild relatives by cross hybridization. In the case of wheat (Triticum aestivum), the species of genus Aegilops are a particularly attractive source of new genes and alleles. However, during the evolution of the Aegilops and Triticum genera, diversification of the D-genome lineage resulted in the formation of diploid C, M, and U genomes of Aegilops. The extent of structural genome alterations, which accompanied their evolution and speciation, and the shortage of molecular tools to detect Aegilops chromatin hamper gene transfer into wheat. To investigate the chromosome structure and help develop molecular markers with a known physical position that could improve the efficiency of the selection of desired introgressions, we developed single-gene fluorescence in situ hybridization (FISH) maps for M- and U-genome progenitors, Aegilops comosa and Aegilops umbellulata, respectively. Forty-three ortholog genes were located on 47 loci in Ae. comosa and on 52 loci in Ae. umbellulata using wheat cDNA probes. The results obtained showed that M-genome chromosomes preserved collinearity with those of wheat, excluding 2 and 6M containing an intrachromosomal rearrangement and paracentric inversion of 6ML, respectively. While Ae. umbellulata chromosomes 1, 3, and 5U maintained collinearity with wheat, structural reorganizations in 2, 4, 6, and 7U suggested a similarity with the C genome of Aegilops markgrafii. To develop molecular markers with exact physical positions on chromosomes of Aegilops, the single-gene FISH data were validated in silico using DNA sequence assemblies from flow-sorted M- and U-genome chromosomes. The sequence similarity search of cDNA sequences confirmed 44 out of the 47 single-gene loci in Ae. comosa and 40 of the 52 map positions in Ae. umbellulata. Polymorphic regions, thus, identified enabled the development of molecular markers, which were PCR validated using wheat-Aegilops disomic chromosome addition lines. The single-gene FISH-based approach allowed the development of PCR markers specific for cytogenetically mapped positions on Aegilops chromosomes, substituting as yet unavailable segregating map. The new knowledge and resources will support the efforts for the introgression of Aegilops genes into wheat and their cloning.

Development of Novel Wheat-Aegilops longissima 3S1 Translocations Conferring Powdery Mildew Resistance and Specific Molecular Markers for Chromosome 3S1.

Powdery mildew of wheat, caused by Blumeria graminis f. sp. tritici, is a destructive disease of common wheat. Cultivation of resistant varieties is the most cost-effective disease management strategy. Previous studies reported that chromosome 3Sl#2 present in Chinese Spring (CS)-Aegilops longissima 3Sl#2(3B) disomic substitution line TA3575 conferred resistance to powdery mildew. In this study, we further located the powdery mildew resistance gene(s) to the short arm of chromosome 3Sl#2 (3Sl#2S) by evaluating for B. graminis f. sp. tritici resistance of newly developed CS-Ae. longissima 3Sl#2 translocation lines. Meanwhile, TA7545, a previously designated CS-Ae. longissima 3Sl#3 disomic addition line, was reidentified as an isochromosome 3Sl#3S addition line and evaluated to confer resistance to powdery mildew, thus locating the resistance gene(s) to the short arm of chromosome 3Sl#3 (3Sl#3S). Based on transcriptome sequences of TA3575, 10 novel chromosome 3SlS-specific markers were developed, of which 5 could be used to distinguish between 3Sl#2S and 3Sl#3S derived from Ae. longissima accessions TL20 and TA1910 (TAM4) and the remaining 5 could identify both 3Sl#2S and 3Sl#3S. Also, CL897, one of five markers specific to both 3Sl#2S and 3Sl#3S, could be used to detect Pm13 located at chromosome 3Sl#1S from Ae. longissima accession TL01 in diverse wheat genetic backgrounds. The powdery mildew resistance genes on chromosomes 3Sl#2S and 3Sl#3S, the CS-Ae. longissima 3Sl#2 translocation lines, and the 3SlS-specific markers developed in this study will facilitate the transfer of B. graminis f. sp. tritici resistance genes into common wheat and provide new germplasm resources for powdery mildew resistance breeding.

Molecular cytogenetic characterization and fusarium head blight resistance of five wheat-Thinopyrum intermedium partial amphiploids.

BACKGROUND: Partial amphiploids created by crossing octoploid tritelytrigia(2n = 8× = 56, AABBDDEE) and Thinopyrum intermedium (2n = 6× = 42, StStJJJSJS) are important intermediates in wheat breeding because of their resistance to major wheat diseases. We examined the chromosome compositions of five wheat-Th. intermedium partial amphiploids using GISH and multicolor-FISH. RESULTS: The result revealed that five lines had 10-14 J-genome chromosomes from Th. intermedium and 42 common wheat chromosomes, using the J-genomic DNA from Th. bessarabicum as GISH probe and the oligo probes pAs1-1, pAs1-3, AFA-4, (GAA) 10, and pSc119.2-1 as FISH probe. Five lines resembled their parent octoploid tritelytrigia (2n = 8× = 56, AABBDDEE) but had higher protein contents. Protein contents of two lines HS2-2 and HS2-5 were up to more than 20%. Evaluation of Fusarium head blight (FHB) resistance revealed that the percent of symptomatic spikelets (PSS) of these lines were below 30%. Lines HS2-2, HS2-4, HS2-5, and HS2-16 were less than 20% of PPS. Line HS2-5 with 14 J-genome chromosomes from Th. intermedium showed the best disease resistance, with PSS values of 10.8% and 16.6% in 2016 and 2017, respectively. CONCLUSIONS: New wheat-Th. intermedium amphiploids with the J-genome chromosomes were identified and can be considered as a valuable source of FHB resistance in wheat breeding.

Deciphering the Mechanism of Glyphosate Resistance in Amaranthus palmeri by Cytogenomics.

In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in Amaranthus palmeri (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to >160-fold increase in copies of the EPSPS gene than in a glyphosate-susceptible (GS) population. This increased copy number of the EPSPS gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb EPSPS cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified EPSPS copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The EPSPS gene-containing eccDNA having a size of ∼400 kb is termed EPSPS-eccDNA and showed somatic mosacism in size and copy number. EPSPS-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the EPSPS locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of EPSPS-eccDNA sheds light on various characteristics of EPSPS-eccDNA that favor GR in AP.

The Aegilops ventricosa 2NvS segment in bread wheat: cytology, genomics and breeding.

KEY MESSAGE: The first cytological characterization of the 2NvS segment in hexaploid wheat; complete de novo assembly and annotation of 2NvS segment; 2NvS frequency is increasing 2NvS and is associated with higher yield. The Aegilops ventricosa 2NvS translocation segment has been utilized in breeding disease-resistant wheat crops since the early 1990s. This segment is known to possess several important resistance genes against multiple wheat diseases including root knot nematode, stripe rust, leaf rust and stem rust. More recently, this segment has been associated with resistance to wheat blast, an emerging and devastating wheat disease in South America and Asia. To date, full characterization of the segment including its size, gene content and its association with grain yield is lacking. Here, we present a complete cytological and physical characterization of this agronomically important translocation in bread wheat. We de novo assembled the 2NvS segment in two wheat varieties, 'Jagger' and 'CDC Stanley,' and delineated the segment to be approximately 33 Mb. A total of 535 high-confidence genes were annotated within the 2NvS region, with > 10% belonging to the nucleotide-binding leucine-rich repeat (NLR) gene families. Identification of groups of NLR genes that are potentially N genome-specific and expressed in specific tissues can fast-track testing of candidate genes playing roles in various disease resistances. We also show the increasing frequency of 2NvS among spring and winter wheat breeding programs over two and a half decades, and the positive impact of 2NvS on wheat grain yield based on historical datasets. The significance of the 2NvS segment in wheat breeding due to resistance to multiple diseases and a positive impact on yield highlights the importance of understanding and characterizing the wheat pan-genome for better insights into molecular breeding for wheat improvement.

Development and Molecular Cytogenetic Characterization of Cold-Hardy Perennial Wheatgrass Adapted to Northeastern China.

Cold-hardy perennial wheatgrass plays an important role in the use of barren land for farming, soil and water conservation, variety improvement, and also for increasing grass yield. By crossing octoploid tritelytrigia (2n = 8x = 56, AABBDDEE) with Thinopyrum intermedium (2n = 6x = 42, StStJJJ S J S ), we developed 34 lines of perennial wheatgrass from F1 to F6 generations, which had vigorous regrowth and cold hardiness. The cold-hardy, perennial wheatgrass lines were well-adapted to the cold environment and developed root and rhizomes, with a longevity between 5 and 11 years and a better seed set. Some of them maintained wheat chromosomes beneficial for breeding perennial wheat. Molecular cytogenetic analysis demonstrated that the Th. intermedium chromosomes contributed the most to the synthetic genome of the wheatgrass hybrids and were associated with the perennial growth habit and winter hardiness. They were also preferentially maintained and transmitted to the progenies. Some wheat chromosomes were also transmitted from the F1 to F6 generations, although they were eliminated in each life cycle of the wheatgrass hybrids. The numbers of wheat and Th. intermedium chromosomes affected seed set and perennial growth habit. Seed set increased with the establishment of a more balanced genomic constitution in later generations. The cold-hardy and perennial wheatgrass lines were produced, which can be the starting point of domestication effort aimed at producing well-adapted ground cover plants under extreme environments.

In-silico detection of aneuploidy and chromosomal deletions in wheat using genotyping-by-sequencing.

BACKGROUND: Short read sequencing technologies, such as genotyping-by-sequencing (GBS), have been utilized in genetic mapping, marker development, and population genomic studies. High-throughput and multiplexing capability coupled with low cost make GBS an appropriate tool for molecular research. Here, we present the application of GBS to characterize wheat aneuploid stocks and detect chromosomal aberrations including aneuploidy and chromosomal deletions. These aneuploids are an important resource that have been used in wheat genetics and genomics studies to localize genes, determine physical positions, and develop chromosome bin maps. RESULTS: Using GBS, we mapped sequence reads and quantified read coverage across chromosome bins. Using this approach, we confirmed known deletions and aneuploid stocks. In addition, we were also able to fully characterize these stocks and to identify several novel deletions and aneuploids. With this knowledge and a quick detection tool at our disposal, we can easily isolate these deletions and aneuploids into distinct lines. CONCLUSION: We envision this tool to replace the intensive cytogenetics techniques, such as C-banding, and fluorescent- and genomic-in situ hybridization to accurately detect chromosome dosage and segmental deletions in wheat genetic stocks as well as other crop species.

Genome-wide impacts of alien chromatin introgression on wheat gene transcriptions.

Agronomic characteristics and tolerance to biotic and abiotic stresses in hexaploid wheat can be drastically improved through wheat-alien introgression. However, the transcriptional level interactions of introduced alien genes in the wheat genetic background is rarely investigated. In this study, we report the genome-wide impacts of introgressed chromosomes derived from Ae. longissima on gene transcriptions of the wheat landrace Chinese Spring. RNA-seq analyses demonstrated 5.37% and 4.30% of the genes were significantly differentially expressed (DEGs) in CS-Ae. longissima disomic 3Sl#2(3B) substitution line TA3575 and disomic 6Sl#3 addition line TA7548, respectively when compared to CS. In addition, 561 DEGs, including 413 up-regulated and 148 down-regulated or not transcribed genes, were simultaneously impacted by introgressed chromosomes 3Sl#2 and 6Sl#3, which accounts for 41.25% of the DEGs in TA3575 and 38.79% in TA7548. Seventeen DEGs, annotated as R genes, were shared by both introgression lines carrying chromosomes 3Sl#2 and 6Sl#3, which confer resistance to powdery mildew. This study will benefit the understanding of the wheat gene responses as result of alien gene(s) or chromosome intogression and the plant defense response initiated by powdery mildew resistance genes in chromosomes 3Sl#2 and 6Sl#3.

A spontaneous wheat-Aegilops longissima translocation carrying Pm66 confers resistance to powdery mildew.

KEY MESSAGE: A spontaneous Robertsonian T4SlS·4BL translocation chromosome carrying Pm66 for powdery mildew resistance was discovered and confirmed by RNA-seq, molecular marker, and in situ hybridization analyses. Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a severe disease of bread wheat worldwide. Discovery and utilization of resistance genes to powdery mildew from wild relatives of wheat have played important roles in wheat improvement. Aegilops longissima, one of the S-genome diploid wild relatives of wheat, is a valuable source of disease and pest resistance for wheat. Chromosome 4Sl from Ae. longissima confers moderate resistance to powdery mildew. In this study, we conducted RNA-seq on a putative Chinese Spring (CS)-Ae. longissima 4Sl(4B) disomic substitution line (TA3465) to develop 4Sl-specific markers to assist the transfer of a Bgt resistance gene from 4Sl by induced homoeologous recombination. A pairwise comparison of genes between CS and TA3465 demonstrated that a number of genes on chromosome 4BS in CS were not expressed in TA3465. Analysis of 4B- and 4Sl-specific molecular markers showed that 4BS and 4SlL were both missing in TA3465, whereas 4BL and 4SlS were present. Further characterization by genomic and fluorescent in situ hybridization confirmed that TA3465 carried a spontaneous Robertsonian T4SlS·4BL translocation. Powdery mildew tests showed that TA3465 was resistant to 10 of 16 Bgt isolates collected from different regions of China, whereas CS was susceptible to all those Bgt isolates. The powdery mildew resistance gene(s) in TA3465 was further mapped to the short arm of 4Sl and designated as Pm66.

Physical Mapping of Pm57, a Powdery Mildew Resistance Gene Derived from Aegilops searsii.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is one of many severe diseases that threaten bread wheat (Triticum aestivum L.) yield and quality worldwide. The discovery and deployment of powdery mildew resistance genes (Pm) can prevent this disease epidemic in wheat. In a previous study, we transferred the powdery mildew resistance gene Pm57 from Aegilops searsii into common wheat and cytogenetically mapped the gene in a chromosome region with the fraction length (FL) 0.75-0.87, which represents 12% segment of the long arm of chromosome 2Ss#1. In this study, we performed RNA-seq using RNA extracted from leaf samples of three infected and mock-infected wheat-Ae. searsii 2Ss#1 introgression lines at 0, 12, 24, and 48 h after inoculation with Bgt isolates. Then we designed 79 molecular markers based on transcriptome sequences and physically mapped them to Ae. searsii chromosome 2Ss#1- in seven intervals. We used these markers to identify 46 wheat-Ae. searsii 2Ss#1 recombinants induced by ph1b, a deletion mutant of pairing homologous (Ph) genes. After analyzing the 46 ph1b-induced 2Ss#1L recombinants in the region where Pm57 is located with different Bgt-responses, we physically mapped Pm57 gene on the long arm of 2Ss#1 in a 5.13 Mb genomic region, which was flanked by markers X67593 (773.72 Mb) and X62492 (778.85 Mb). By comparative synteny analysis of the corresponding region on chromosome 2B in Chinese Spring (T. aestivum L.) with other model species, we identified ten genes that are putative plant defense-related (R) genes which includes six coiled-coil nucleotide-binding site-leucine-rich repeat (CNL), three nucleotide-binding site-leucine-rich repeat (NL) and a leucine-rich receptor-like repeat (RLP) encoding proteins. This study will lay a foundation for cloning of Pm57, and benefit the understanding of interactions between resistance genes of wheat and powdery mildew pathogens.

Physical Mapping of Stem Rust Resistance Gene Sr52 from Dasypyrum villosum Based on ph1b-Induced Homoeologous Recombination.

Wheat stem rust caused by Puccinia graminis f. sp. tritici (Pgt) had been a devastating foliar disease worldwide during the 20th century. With the emergence of Ug99 races, which are virulent to most stem rust resistance genes deployed in wheat varieties and advanced lines, stem rust has once again become a disease threatening global wheat production. Sr52, derived from Dasypyrum villosum and mapped to the long arm of 6V#3, is one of the few effective genes against Ug99 races. In this study, the wheat-D. villosum Robertsonian translocation T6AS·6V#3L, the only stock carrying Sr52 released to experimental and breeding programs so far, was crossed with a CS ph1b mutant to induce recombinants with shortened 6V#3L chromosome segments locating Sr52. Six independent homozygous recombinants with different segment sizes and breakpoints were developed and characterized using in situ hybridization and molecular markers analyses. Stem rust resistance evaluation showed that only three terminal recombinants (1381, 1380, and 1392) containing 8%, 22%, and 30% of the distal segment of 6V#3L, respectively, were resistant to stem rust. Thus, the gene Sr52 was mapped into 6V#3L bin FL 0.92-1.00. In addition, three molecular markers in the Sr52-located interval of 6V#3L were confirmed to be diagnostic markers for selection of Sr52 introgressed into common wheat. The newly developed small segment translocation lines with Sr52 and the identified molecular markers closely linked to Sr52 will be valuable for wheat disease breeding.

Production of a complete set of wheat-barley group-7 chromosome recombinants with increased grain ß-glucan content.

KEY MESSAGE: Wheat-barley group-7 recombinant chromosomes were selected using molecular cytogenetics and SNP markers; increased grain ß-glucan content was observed in wheat plants with two and four copies of HvCslF6. The soluble dietary fiber (1-3)(1-4) mixed linked ß-D-glucan from cereal grains is a valuable component of a healthy diet, which reduces risks of coronary disease and diabetes. Although wheat is an important cereal crop providing a substantial portion of daily calories and protein intake in the human diet, it has a low level of ß-glucan. Owing to the plasticity of the polyploid wheat genome, agronomically important traits absent in the wheat primary gene pool can be introgressed from distant relatives. Barley (Hordeum vulgare L.) has a high grain ß-glucan content. Earlier, we introgressed this trait into wheat in the form of whole arm compensating Robertsonian translocations (RobT) involving group-7 chromosomes of barley and all three sub-genomes of hexaploid wheat (Triticum aestivum L). In the presented research, we shortened the barley 7HL arms in these RobTs to small pericentromeric segments, using induced wheat-barley homoeologous recombination. The recombinants were selected using SNP markers and molecular cytogenetics. Plants, comprising barley cellulose synthase-like F6 gene (HvCslF6), responsible for ß-glucan synthesis, had a higher grain ß-glucan content than the wheat control. Three wheat-barley group-7 recombinant chromosomes involving the A, B and D sub-genomes laid the basis for a multiple-copy gene introgression to hexaploid wheat. It is hypothesized that further increases in the ß-glucan content in wheat grain can be obtained by increasing the number of HvCslF6 copies through combining several recombinant chromosomes in one line. The wheat lines with four copies of HvCslF6 exceeded the ß-glucan content of the lines with two copies.