Characterisation of Thinopyrum bessarabicum chromosomes through genome-wide introgressions into wheat

Thinopyrum bessarabicum (2n = 2x = 14, JJ) is an important source for new genetic variation for wheat improvement due to its salinity tolerance and disease resistance. Its practical utilisation in wheat improvement can be facilitated through development of genome-wide introgressions leading to a variety of different wheat–Th. bessarabicum translocation lines. In this study, we report the generation of 12 such wheat–Th. bessarabicum recombinant lines, through two different crossing strategies, which were characterized using sequential chromosome single colour and multi-colour genomic in situ hybridization (sc-GISH and mc-GISH), multi-colour fluorescent in situ hybridization (mc-FISH) and single nucleotide polymorphic (SNP) DNA markers. We also detected 13 lines containing different Th. bessarabicum chromosome aberrations through sc-GISH. Through a combination of molecular and cytological analysis of all the 25 lines containing Th. bessarabicum recombinants and chromosome aberrations we were able to physically map 1150 SNP markers onto seven Th. bessarabicum J chromosomes which were divided into 36 segmental blocks. Comparative analysis of the physical map of Th. bessarabicum and the wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed that Th. bessarabicum contains the 4/5 translocation also present in the A genome of wheat. These wheat–Th. bessarabicum recombinant lines and SNP markers provide a useful genetic resource for wheat improvement with the latter having a wider impact as a tool for detection of introgressions from other Thinopyrum species containing the J or a closely-related genome such as Thinopyrum intermedium (JrJrJvsJvsStSt) and Thinopyrum elongatum (EeEe), respectively

Detection of T. urartu Introgressions in Wheat and Development of a Panel of Interspecific Introgression Lines

Tritcum urartu (2n = 2x = 14, AuAu), the A genome donor of wheat, is an important source for new genetic variation for wheat improvement due to its high photosynthetic rate and disease resistance. By facilitating the generation of genome-wide introgressions leading to a variety of different wheat–T. urartu translocation lines, T. urartu can be practically utilised in wheat improvement. Previous studies that have generated such introgression lines have been unable to successfully use cytological methods to detect the presence of T. urartu in these lines. Many have, thus, used a variety of molecular markers with limited success due to the low-density coverage of these markers and time-consuming nature of the techniques rendering them unsuitable for large-scale breeding programs. In this study, we report the generation of a resource of single nucleotide polymorphic (SNP) markers, present on a high-throughput SNP genotyping array, that can detect the presence of T. urartu in a hexaploid wheat background making it a potentially valuable tool in wheat pre-breeding programs. A whole genome introgression approach has resulted in the transfer of different chromosome segments from T. urartu into wheat which have then been detected and characterised using these SNP markers. The molecular analysis of these wheat-T. urartu recombinant lines has resulted in the generation of a genetic map of T. urartu containing 368 SNP markers, spread across all seven chromosomes of T. urartu. Comparative analysis of the genetic map of T. urartu and the physical map of the hexaploid wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed the presence of the 4/5 translocation in T. urartu also present in the A genome of wheat. A panel of 17 wheat-T. urartu recombinant lines, which consisted of introgressed segments that covered the whole genome of T. urartu, were also selected for self-fertilisation to provide a germplasm resource for future trait analysis. This valuable resource of high-density molecular markers specifically designed for detecting wild relative chromosomes and a panel of stable interspecific introgression lines will greatly enhance the efficiency of wheat improvement through wild relative introgressions

Development of a New A –Genome-Specific Single Nucleotide Polymorphism Marker Set for the Molecular Characterization of Wheat– Introgression Lines

© 2019 The Author(s). Cultivated einkorn wheat (Triticum monococcum L. subsp. monococcum, 2n = 2x = 14, AmAm) and its wild relative T. monococcum subsp. aegilopoides are important sources of economically useful genes that can be exploited for wheat (Triticum aestivum L.) breeding. Einkorn has excellent resistance to fungal diseases and gene transfer is relatively simple via standard breeding methods. To fulfill the growing demand by modern prebreeding programs for a cost-effective high-throughpuprocedure for accurately detecting introgressed chromosomes or chromosome segments from T. monococcum into wheat, we used the Axiom Wheat-Relative Genotyping Array and developed a set of Am genome-specific exome-based single nucleotide polymorphism (SNP) markers suitable for rapid identification of T. monococcum chromatin in a wheat background. We identified 1247 polymorphic SNPs between T. monococcum and wheat. We identified 191 markers across all seven chromosomes of T. monococcum that are also present on an existing Triticum urartu Thum. ex Gandil. genetic map and potentially ordered them on the basis of the high macrocollinearity and conservation of marker order between T. monococcum and T. urartu. The marker set has been tested on leaf-rust-resistant BC3 F4 progenies of wheat–T. monococcum hybrids. Two markers (AX-94492165, AX-95073542) placed on the distal end of the chromosome arm 7AL detected a T. monococcum introgression into wheat. The SNP marker set thus proved highly effective in the identification of T. monococcum chromatin in a wheat background, offering a reliable method for screening and selecting wheat–T. monococcum introgression lines, a procedure that could significantly speed up prebreeding programs

Development and characterisation of interspecific hybrid lines with genome-wide introgressions from <i>Triticum timopheevii</i> in a hexaploid wheat background

© 2019 The Author(s). Background: Triticum timopheevii (2n = 4x = 28; AtAtGG), is an important source for new genetic variation for wheat improvement with genes for potential disease resistance and salt tolerance. By generating a range of interspecific hybrid lines, T. Timopheevii can contribute to wheat's narrow gene-pool and be practically utilised in wheat breeding programmes. Previous studies that have generated such introgression lines between wheat and its wild relatives have been unable to use high-throughput methods to detect the presence of wild relative segments in such lines. Results: A whole genome introgression approach, exploiting homoeologous recombination in the absence of the Ph1 locus, has resulted in the transfer of different chromosome segments from both the At and G genomes of T. Timopheevii into wheat. These introgressions have been detected and characterised using single nucleotide polymorphism (SNP) markers present on a high-throughput Axiom® Genotyping Array. The analysis of these interspecific hybrid lines has resulted in the detection of 276 putative unique introgressions from T. Timopheevii, thereby allowing the generation of a genetic map of T. Timopheevii containing 1582 SNP markers, spread across 14 linkage groups representing each of the seven chromosomes of the At and G genomes of T. Timopheevii. The genotyping of the hybrid lines was validated through fluorescence in situ hybridisation (FISH). Comparative analysis of the genetic map of T. Timopheevii and the physical map of the hexaploid wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed the presence of inter- and intra-genomic translocations within the At and G genomes of T. Timopheevii that have been previously only detected through cytological techniques. Conclusions: In this work, we report a set of SNP markers present on a high-throughput genotyping array, able to detect the presence of T. Timopheevii in a hexaploid wheat background making it a potentially valuable tool for marker assisted selection (MAS) in wheat pre-breeding programs. These valuable resources of high-density molecular markers and wheat-T. Timopheevii hybrid lines will greatly enhance the work being undertaken for wheat improvement through wild relative introgressions

Development of Wheat-Aegilops caudata Introgression Lines and Their Characterization Using Genome-Specific KASP Markers

© Copyright © 2020 Grewal, Othmeni, Walker, Hubbart-Edwards, Yang, Scholefield, Ashling, Isaac, King and King. Aegilops caudata L. [syn. Ae. markgrafii (Greuter) Hammer], is a diploid wild relative of wheat (2n = 2x = 14, CC) and a valuable source for new genetic diversity for wheat improvement. It has a variety of disease resistance factors along with tolerance for various abiotic stresses and can be used for wheat improvement through the generation of genome-wide introgressions resulting in different wheat–Ae. caudata recombinant lines. Here, we report the generation of nine such wheat–Ae. caudata recombinant lines which were characterized using wheat genome-specific KASP (Kompetitive Allele Specific PCR) markers and multi-color genomic in situ hybridization (mcGISH). Of these, six lines have stable homozygous introgressions from Ae. caudata and will be used for future trait analysis. Using cytological techniques and molecular marker analysis of the recombinant lines, 182 KASP markers were physically mapped onto the seven Ae. caudata chromosomes, of which 155 were polymorphic specifically with only one wheat subgenome. Comparative analysis of the physical positions of these markers in the Ae. caudata and wheat genomes confirmed that the former had chromosomal rearrangements with respect to wheat, as previously reported. These wheat–Ae. caudata recombinant lines and KASP markers are useful resources that can be used in breeding programs worldwide for wheat improvement. Additionally, the genome-specific KASP markers could prove to be a valuable tool for the rapid detection and marker-assisted selection of other Aegilops species in a wheat background

Development of stable homozygous wheat/Amblyopyrum muticum (Aegilops mutica) introgression lines and their cytogenetic and molecular characterization

Wheat is one of the world’s most important sources of food. However, due to its evolution its genetic base has narrowed, which is severely limiting the ability of breeders to develop new higher yielding varieties that can adapt to the changing environment. In contrast to wheat, its wild relatives provide a vast reservoir of genetic variability for most, if not all, agronomically important traits. Genetic variation has previously been transferred to wheat from one of its wild relatives, Ambylopyrum muticum (previously known as Aegilops mutica). However, before the genetic variation available in this species can be assessed and exploited in breeding and for research, the transmission of the chromosome segments introgressed into wheat must first be stabilized. In this paper we describe the generation of 66 stably inherited homozygous wheat/Am. muticum introgression lines using a doubled haploid procedure. The characterisation and stability of each of these lines was determined via genomic in situ hybridization and SNP analysis. While most of the doubled haploid lines were found to carry only single introgressions, six lines carried two. Three lines carried only complete Am. muticum chromosomes, 43 carried only small or very small introgressions and the remainder carried either only large introgressions or a large plus a small introgression. The strategy that we are employing for the distribution and exploitation of the genetic variation from Am. muticum and a range of other species is discussed

Chromosome-specific KASP markers for detecting Amblyopyrum muticum segments in wheat introgression lines

Many wild-relative species are being used in prebreeding programs to increase the genetic diversity of wheat (Triticum aestivum L.). Genotyping tools such as single nucleotide polymorphism (SNP)-based arrays and molecular markers have been widely used to characterize wheat–wild relative introgression lines. However, due to the polyploid nature of the recipient wheat genome, it is difficult to develop SNP-based Kompetitive allele-specific polymerase chain reaction (KASP) markers that are codominant to track the introgressions from the wild species. Previous attempts to develop KASP markers have involved both exome- and polymerase chain reaction (PCR)-amplicon-based sequencing of the wild species. But chromosome-specific KASP assays have been hindered by homoeologous SNPs within the wheat genome. This study involved whole genome sequencing of the diploid wheat wild relative Amblyopyrum muticum (Boiss.) Eig and development of a de novo SNP discovery pipeline that generated ∼38,000 SNPs in unique wheat genome sequences. New assays were designed to increase the density of Am. muticum polymorphic KASP markers. With a goal of one marker per 60 Mbp, 335 new KASP assays were validated as diagnostic for Am. muticum in a wheat background. Together with assays validated in previous studies, 498 well distributed chromosome-specific markers were used to recharacterize previously genotyped wheat–Am. muticum doubled haploid (DH) introgression lines. The chromosome-specific nature of the KASP markers allowed clarification of which wheat chromosomes were involved with recombination events or substituted with Am. muticum chromosomes and the higher density of markers allowed detection of new small introgressions in these DH lines

Development and validation of an exome-based SNP marker set for identification of the St, J ^r and J ^vs genomes of Thinopyrym intermedium in a wheat background

Thinopyrum intermedium (2n = 6x = 42, StJrJvs) is one of the most promising reservoirs of useful genes including tolerance to abiotic stresses, perenniality and disease resistance not available in the cultivated bread wheat. The transfer of genetic diversity from wild species to wheat offers valuable responses to the effects of climate change. The new array-based single-nucleotide polymorphism (SNP) marker technology provides cheap and easy-to-use molecular markers for marker-assisted selection (MAS) in wheat breeding programmes. Here, we focus on the generation of a new chromosome-specific SNP marker set that can be used to characterize and identify the Th. intermedium chromosomes or chromosome segments transferred into wheat. A progressive investigation of marker development was conducted using 187 various newly developed wheat–Th. intermedium introgression lines and the Axiom® Wheat-Relative Genotyping array. We employed molecular cytogenetic techniques to clarify the genome constitution of the Th. intermedium parental lines and validated 634 chromosome-specific SNPs. Our data confirmed the allohexaploid nature of Th. intermedium and demonstrated that the St genome-specific GISH signal and markers are present at the centromeric regions of chromosomes 1Jvs, 2Jvs, 3Jvs and 7Jvs. The SNP markers presented here will be introduced into current wheat improvement programmes, offering a significant speed-up in wheat breeding and making it possible to deal with the transfer of the full genetic potential of Th. intermedium into wheat

The use of pentaploid crosses for the introgression of Amblyopyrum muticum and D-genome chromosome segments into durum wheat

The wild relatives of wheat provide an important source of genetic variation for wheat improvement. Much of the work in the past aimed at transferring genetic variation from wild relatives into wheat has relied on the exploitation of the ph1b mutant, located on the long arm of chromosome 5B. This mutation allows homoeologous recombination to occur between chromosomes from related but different genomes, e.g. between the chromosomes of wheat and related chromosomes from a wild relative resulting in the generation of interspecific recombinant chromosomes. However, the ph1b mutant also enables recombination to occur between the homoeologous genomes of wheat, e.g. A/B, A/D, B/D, resulting in the generation of wheat intergenomic recombinant chromosomes. In this work we report on the presence of wheat intergenomic recombinants in the genomic background of hexaploid wheat/Amblyopyrum muticum introgression lines. The transfer of genomic rearrangements involving the D-genome through pentaploid crosses provides a strategy by which the D-genome of wheat can be introgressed into durum wheat. Hence, a pentaploid crossing strategy was used to transfer D-genome segments, translocated with either the A- and/or the B-genome, into the tetraploid background of two durum wheat genotypes Karim and Om Rabi 5 in either the presence or absence of different Am. muticum (2n = 2x = 14, TT) segments. Translocations were monitored in backcross generations to the durum wheat parents via multicolour genomic in situ hybridisation (mc-GISH). Tetraploid lines carrying homozygous D-genome introgressions, as well as simultaneous homozygous D- and T-genome introgressions, were developed. Introgression lines were characterised via KASP markers and multicolour fluorescence in situ hybridisation (FISH). Results showed that new wheat sub-genomic translocations were generated at each generation in progeny that carried any Am. muticum chromosome segment irrespective of the linkage group that the segment was derived from. The highest frequencies of homoeologous recombination were observed between the A- and the D-genomes. Results indicated that the genotype Karim had a higher tolerance to genomic rearrangements and T-genome introgressions compared to Om Rabi 5. This indicates the importance of the selection of the parental genotype when attempting to transfer/develop introgressions into durum wheat from pentaploid crosses

Generation of Doubled Haploid Wheat-Triticum urartu Introgression Lines and Their Characterisation Using Chromosome-Specific KASP Markers

Wheat is one of the most important food and protein sources in the world and although, in recent years wheat breeders have achieved yield gains, they are not sufficient to meet the demands of an ever-growing population. Development of high yielding wheat varieties, resilient to abiotic and biotic stress resulting from climate change, has been limited by wheat’s narrow genetic base. In contrast to wheat, the wild relatives of wheat provide a vast reservoir of genetic variation for most, if not all, agronomic traits. Previous studies by the authors have shown the transfer of genetic variation from T. urartu into bread wheat. However, before the introgression lines can be exploited for trait analysis, they are required to have stable transmission of the introgressions to the next generation. In this work, we describe the generation of 86 doubled haploid (DH) wheat-T. urartu introgression lines that carry homozygous introgressions which are stably inherited. The DH lines were characterised using the Axiom® Wheat Relative Genotyping Array and 151 KASP markers to identify 65 unique T. urartu introgressions in a bread wheat background. DH production has helped accelerate the breeding process and facilitated the early release of homozygous wheat-T. urartu introgression lines. Together with the KASP markers, this valuable resource could greatly advance identification of beneficial alleles that can be used in wheat improvement