GenoBaits®WheatplusEE: a targeted capture sequencing panel for quick and accurate identification of wheat-Thinopyrum derivatives.

KEY MESSAGE: A total of 90,000 capture probes derived from wheat and Thinopyrum elongatum were integrated into one chip, which served as an economical genotype for explorating Thinopyrumspecies and their derivatives. Thinopyrum species play a crucial role as a source of new genetic variations for enhancing wheat traits, including resistance to both abiotic and biotic factors. Accurate identification of exogenous chromosome(s) or chromosome segments or genes is essential following the introduction of alien genetic material into wheat, but this task remains challenging. This study aimed to develop a high-resolution wheat-Thinopyrum elongatum array, named GenoBaits®WheatplusEE, to trace alien genetic information by genotyping using a target sequencing system. This GenoBaits®WheatplusEE array included 90,000 capture probes derived from two species and integrated into one chip, with 10,000 and 80,000 originating from wheat and Th. elongatum, respectively. The capture probes were strategically positioned in genes and evenly distributed across the genome, facilitating the development of a roadmap for identifying each alien gene. The array was applied to the high-throughput identification of the alien chromosomes or segments in Thinopyrum and distantly related species and their derivatives. Our results demonstrated that the GenoBaits®WheatplusEE array could be used for direct identification of the breakpoint of alien segments, determine copy number of alien chromosomes, and reveal variations in wheat chromosomes by a single round of target sequencing of the sample. Additionally, we could efficiently and cost-effectively genotype, supporting the exploration of subgenome composition, phylogenetic relationships, and polymorphisms in essential genes (e.g., Fhb7 gene) among Thinopyrum species and their derivatives. We hope that GenoBaits®WheatplusEE will become a widely adopted tool for exporting wild germplasm for wheat improvement 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.

Identification and DNA Marker Development for a Wheat-Leymus mollis 2Ns (2D) Disomic Chromosome Substitution.

Leymus mollis (2n = 4x = 28, NsNsXmXm), a wild relative of common wheat (Triticum aestivum L.), carries numerous loci which could potentially be used in wheat improvement. In this study, line 17DM48 was isolated from the progeny of a wheat and L. mollis hybrid. This line has 42 chromosomes forming 21 bivalents at meiotic metaphase I. Genomic in situ hybridization (GISH) demonstrated the presence of a pair chromosomes from the Ns genome of L. mollis. This pair substituted for wheat chromosome 2D, as shown by fluorescence in situ hybridization (FISH), DNA marker analysis, and hybridization to wheat 55K SNP array. Therefore, 17DM48 is a wheat-L. mollis 2Ns (2D) disomic substitution line. It shows longer spike and a high level of stripe rust resistance. Using specific-locus amplified fragment sequencing (SLAF-seq), 13 DNA markers were developed to identify and trace chromosome 2Ns of L. mollis in wheat background. This line provides a potential bridge germplasm for genetic improvement of wheat stripe rust resistance.

Chromosome karyotype and stability of new synthetic hexaploid wheat.

Synthetic hexaploid wheat offers breeders ready access to potentially novel genetic variation in wild ancestral species. In this study, we crossed MY3478 (2n = 4x = 28, AABB) as the maternal parent with the stripe rust-resistant SY41 (2n = 2x = 14, DD) as the paternal parent to construct the new hexaploid wheat line NA0928 through natural allopolyploidization. Agronomic traits and the cytology of the S8-S9 generations of NA0928 were analyzed. Abundant variation in agronomic traits was observed among each strain of NA0928 in the S8 generation. Agronomic traits were superior in strains resistant to stripe rust compared with those of highly susceptible strains. The rank order of the coefficients of variation were tiller number (55.3%) > spike length (15.3%) > number of spikelets (13.9%) > plant height (8.7). Number of tillers and spike length are important traits in wheat breeding to improve yield. Cytological observation and fluorescence in situ hybridization showed that the chromosome number and configuration showed rich variation among NA0928 strains in the S9 generation. Chromosome number ranged from 36 to 44. Variation in chromosome karyotype was detected in the A and B subgenomes. Meiotic chromosome behavior in pollen mother cells and multicolor genomic in situ hybridization revealed that two new synthetic hexaploid wheat strains showed genetic stability; one strain was resistant to stripe rust and developed multiple tillers, and the other strain was susceptible to stripe rust, but both showed improved thousand-kernel weight (TKW) weight and produced multiple tillers. The two strains will be valuable germplasm resources for use in wheat breeding.

Gene co-expression network analysis provides a novel insight into the dynamic response of wheat to powdery mildew stress.

Powdery mildew (Blumeria graminis f. sp. Tritici, (Bgt)) is an important worldwide fungal foliar disease of wheat (Triticum aestivum) responsible for severe yield losses. The development of resistance genes and dissection of the resistance mechanism will therefore be beneficial in wheat breeding. The Bgt resistance gene PmAS846 was transferred to the hexaploid wheat lines N9134 from Triticum dicoccoides, and it is still one of the most effective resistance genes. Here, by RNA sequencing, we identified three co-expressed gene modules using pairwise comparisons and weighted gene co-expression network analysis during wheat-Bgt interactions compared with mock-infected plants. Hub genes of stress-specific modules were significantly enriched in spliceosomes, phagosomes, the mRNA surveillance pathway, protein processing in the endoplasmic reticulum, and endocytosis. Induced module genes located on chromosome 5BL were selected to construct a protein-protein interaction network. Several proteins were predicted as the key hub node, including Hsp70, DEAD/DEAH box RNA helicase PRH75, elongation factor EF-2, cell division cycle 5, ARF guanine-nucleotide exchange factor GNOM-like, and protein phosphatase 2C 70 protein, which interacted with several disease resistance proteins such as RLP37, RPP13 and RPS2 analogues. Gene ontology enrichment results showed that wheat could activate binding functional genes via an mRNA transcription mechanism in response to Bgt stress. Of these node genes, GNOM-like, PP2C isoform X1 and transmembrane 9 superfamily member 9 were mapped onto the genetic fragment of PmAS846 with a distance of 4.8 Mb. This work provides the foundations for understanding the resistance mechanism and cloning the resistance gene PmAS846.

Characterization and Evaluation of Resistance to Powdery Mildew of Wheat-Aegilops geniculata Roth 7Mg (7A) Alien Disomic Substitution Line W16998.

Aegilops geniculata Roth has been used as a donor of disease-resistance genes, to enrich the gene pool for wheat (Triticum aestivum) improvement through distant hybridization. In this study, the wheat-Ae. geniculata alien disomic substitution line W16998 was obtained from the BC1F8 progeny of a cross between the common wheat 'Chinese Spring' (CS) and Ae. geniculata Roth (serial number: SY159//CS). This line was identified using cytogenetic techniques, analysis of genomic in situ hybridization (GISH), functional molecular markers (Expressed sequence tag-sequence-tagged site (EST-STS) and PCR-based landmark unique gene (PLUG), fluorescence in situ hybridization (FISH), sequential fluorescence in situ hybridization-genomic in situ hybridization (sequential FISH-GISH), and assessment of agronomic traits and powdery mildew resistance. During the anaphase of meiosis, these were evenly distributed on both sides of the equatorial plate, and they exhibited high cytological stability during the meiotic metaphase and anaphase. GISH analysis indicated that W16998 contained a pair of Ae. geniculata alien chromosomes and 40 common wheat chromosomes. One EST-STS marker and seven PLUG marker results showed that the introduced chromosomes of Ae. geniculata belonged to homoeologous group 7. Nullisomic-tetrasomic analyses suggested that the common wheat chromosome, 7A, was absent in W16998. FISH and sequential FISH-GISH analyses confirmed that the introduced Ae. geniculata chromosome was 7Mg. Therefore, W16998 was a wheat-Ae. geniculata 7Mg (7A) alien disomic substitution line. Inoculation of isolate E09 (Blumeria graminis f. sp. tritici) in the seedling stage showed that SY159 and W16998 were resistant to powdery mildew, indeed nearly immune, whereas CS was highly susceptible. Compared to CS, W16998 exhibited increased grain weight and more spikelets, and a greater number of superior agronomic traits. Consequently, W16998 was potentially useful. Germplasms transfer new disease-resistance genes and prominent agronomic traits into common wheat, giving the latter some fine properties for breeding.

Isolation and molecular cytogenetic characterization of a wheat - Leymus mollis double monosomic addition line and its progenies with resistance to stripe rust.

A common wheat - Leymus mollis (2n = 4x = 28, NsNsXmXm) double monosomic addition line, M11003-4-3-8/13/15 (2n = 44 = 42T.a + L.m2 + L.m3), with stripe rust resistance was developed (where T.a represents Triticum aestivum chromosome, L.m represents L. mollis chromosome, and L.m2/3 represents L. mollis chromosome of homoeologous groups 2 and 3). The progenies of line M11003-4-3-8/13/15 were characterized by cytological observation, specific molecular markers, fluorescence in situ hybridization (FISH), and genomic in situ hybridization (GISH). Among the progenies, there existed five different types (I, II, III, IV, and V) of chromosome constitution, the formulas of which were 2n = 44 = 42T.a + 1L.m2 + 1L.m3, 2n = 43 = 42T.a + 1L.m2, 2n = 43 = 42T.a + 1L.m3, 2n = 42 = 42T.a, and 2n = 44 = 42T.a + 2L.m2, respectively. Field disease screening showed that types I and III showed high resistance to stripe rust, while types II, IV, and V were susceptible. Leymus mollis was almost immune to stripe rust, whereas the wheat parent, cultivar 7182, was susceptible. Therefore, we concluded that the stripe rust resistance originated from L. mollis. These various lines could be further fully exploited as important disease resistance materials to enrich wheat genetic resources.

Molecular cytogenetics identification of a wheat - Leymus mollis double disomic addition line with stripe rust resistance.

Leymus mollis (Trin.) Pilg. (2n = 4x = 28, NsNsXmXm) possesses a number of valuable genes against biotic and abiotic stress, which could be transferred into common wheat background for wheat improvement. In the present study, we determined the karyotypic constitution of a wheat - L. mollis double disomic addition line, M11003-4-4-1-1, selected from the F5 progeny of a stable wheat - L. mollis derivative M39 (2n = 56) × Triticum aestivum cultivar 7182, by morphological and cytogenetic identification, GISH (genomic in situ hybridization), FISH (fluorescent in situ hybridization), molecular markers analysis, and stripe rust resistance evaluation. Cytological studies demonstrated that M11003-4-4-1-1 had a chromosome karyotype of 2n = 46 with 23 bivalents, while GISH and FISH analysis indicated that this line contained 42 common wheat chromosomes and two pairs of L. mollis chromosomes. DNA markers showed that the alien chromosomes from L. mollis belonged to homoeologous groups 5 and 6. Evaluation of the agronomic traits revealed that M11003-4-4-1-1 was resistant to stripe rust at the adult stage, while the plant height was reduced and the 1000-grain weight was increased significantly. Therefore, the new line M11003-4-4-1-1 could be exploited as an important bridge material in chromosome engineering and wheat breeding.

Development and Molecular Cytogenetic Identification of a Novel Wheat-Leymus mollis Lm#7Ns (7D) Disomic Substitution Line with Stripe Rust Resistance.

Leymus mollis (2n = 4x = 28, NsNsXmXm) possesses novel and important genes for resistance against multi-fungal diseases. The development of new wheat-L. mollis introgression lines is of great significance for wheat disease resistance breeding. M11003-3-1-15-8, a novel disomic substitution line of common wheat cv. 7182 -L. mollis, developed and selected from the BC1F5 progeny between wheat cv. 7182 and octoploid Tritileymus M47 (2n = 8x = 56, AABBDDNsNs), was characterized by morphological and cytogenetic identification, analysis of functional molecular markers, genomic in situ hybridization (GISH), sequential fluorescence in situ hybridization (FISH)-genomic in situ hybridization (GISH) and disease resistance evaluation. Cytological observations suggested that M11003-3-1-15-8 contained 42 chromosomes and formed 21 bivalents at meiotic metaphase I. The GISH investigations showed that line contained 40 wheat chromosomes and a pair of L. mollis chromosomes. EST-STS multiple loci markers and PLUG (PCR-based Landmark Unique Gene) markers confirmed that the introduced L. mollis chromosomes belonged to homoeologous group 7, it was designated as Lm#7Ns. While nulli-tetrasomic and sequential FISH-GISH analysis using the oligonucleotide Oligo-pSc119.2 and Oligo-pTa535 as probes revealed that the wheat 7D chromosomes were absent in M11003-3-1-15-8. Therefore, it was deduced that M11003-3-1-15-8 was a wheat-L. mollis Lm#7Ns (7D) disomic substitution line. Field disease resistance demonstrated that the introduced L. mollis chromosomes Lm#7Ns were responsible for the stripe rust resistance at the adult stage. Moreover, M11003-3-1-15-8 had a superior numbers of florets. The novel disomic substitution line M11003-3-1-15-8, could be exploited as an important genetic material in wheat resistance breeding programs and genetic resources.

Chromosome constitution and origin analysis in three derivatives of Triticum aestivum--Leymus mollis by molecular cytogenetic identification.

Leymus mollis (2n = 4x = 28, NsNsXmXm) is an important tetraploid species in Leymus (Poaceae: Triticeae) and a useful genetic resource for wheat breeding because of the stress tolerance and disease resistance of this species. The development of Triticum aestivum (common wheat) - L. mollis derivatives with desirable genes will provide valuable bridge materials for wheat improvement, especially regarding powdery mildew resistance genes, which are rarely documented in L. mollis. In the present study, three derivatives of common wheat cultivar 7182 and L. mollis, namely M47, M51, and M42, were subjected to chromosomal characterization via cytogenetic identification, the analysis of molecular markers, and genomic in situ hybridization. These derivatives were all morphologically and cytogenetically stable. M47 was highly resistant to powdery mildew and nearly immune to stripe rust at the adult stage, and the chromosome constitution of this derivative can be expressed as 2n = 56 = 42T.a + 14L.m (where T.a = T. aestivum chromosomes; L.m = L. mollis chromosomes). Compared to M47, M42 was also resistant to stripe rust but was susceptible to powdery mildew; the chromosome constitution of M42 was 2n = 54 = 42T.a + 12L.m, in which a pair of homoeologous group 7 L.m chromosomes was eliminated. Finally, M51 was susceptible to powdery mildew and stripe rust and had a chromosome constitution of 2n = 48 = 42T.a + 6L.m, in which four pairs of L.m chromosomes from homoeologous groups 2, 4, 5, and 7 were eliminated. The differing disease resistances of the three derivatives are discussed in this report in the context of their chromosomal variations; this information can thus contribute to breeding disease resistant wheat with the potential for applying these derivatives as useful bridge materials.