Molecular Cytogenetic Identification of Wheat-Aegilops Biuncialis 5Mb Disomic Addition Line with Tenacious and Black Glumes.
ABSTRACT: Production of wheat-alien disomic addition lines is of great value to the exploitation and utilization of elite genes originated from related species to wheat. In this study, a novel wheat-Aegilops biuncialis 5Mb disomic addition line WA317 was characterized by in situ hybridization (ISH) and specific-locus amplified fragment sequencing (SLAF-seq) markers. Compared to its parent Chinese Spring (CS), the glumes of WA317 had black color and were difficult to remove after harvesting, suggesting chromosome 5Mb carried gene(s) related to glume development and Triticeae domestication process. A total of 242 Ae. biuncialis SLAF-based markers (298 amplified patterns) were developed and further divided into four categories by Ae. biuncialis Y17, Ae. umbellulata Y139 and Ae. comosa Y258, including 172 markers amplifying the same bands of U and M genome, six and 102 markers amplifying U-specific and M-specific bands, respectively and eighteen markers amplifying specific bands in Y17. Among them, 45 markers had the specific amplifications in WA317 and were 5Mb specific markers. Taken together, line WA317 with tenacious and black glumes should serve as the foundation for understanding of the Triticeae domestication process and further exploitation of primitive alleles for wheat improvement. Ae. biuncialis SLAF-based markers can be used for studying syntenic relationships between U and M genomes as well as rapid tracking of U and M chromosomal segments in wheat background.
Project description:Powdery mildew is one of the most widespread diseases of wheat. The development and deployment of resistant varieties are one of the most economical and effective methods to manage this disease. Our previous study showed that the gene(s) at 2Mb in Chinese Spring (CS)-Aegilops biuncialis 2Mb disomic addition line TA7733 conferred a high level of resistance to powdery mildew of wheat. In this study, resistance spectrum of TA7733 was assayed by using 15 Blumeria graminis f. sp. tritici (Bgt) isolates prevalent in different regions of China. The result indicated that TA7733 was highly resistant to all tested Bgt isolates and the gene(s) on chromosome 2Mb conferred broad-spectrum resistance to powdery mildew. In order to characterize mechanism of powdery mildew resistance by identifying candidates R-genes derived from Ae. biuncialis chromosome 2Mb and develop 2Mb-specific molecular markers, we performed RNA-seq analysis on TA7733 and CS. In total we identified 7,278 unigenes that showed specific expression in TA7733 pre and post Bgt-infection when compared to CS. Of these 7,278 unigenes, 295 were annotated as putative resistance (R) genes. Comparatively analysis of R-gene sequences from TA7733 and CS and integration CS Ref Seq v1.0 were used to develop R-gene specific primers. Of 295 R-genes we identified 53 R-genes were specific to 2Mb and could be involved in powdery mildew resistance. Functional annotation of majority of the 53 R-genes encoded nucleotide binding leucine rich repeat (NLR) protein. The broad-spectrum resistance to powdery mildew in TA7733 and availability of 2Mb-derived putative candidate R-gene specific molecular markers identified in this study will lay foundations for transferring powdery mildew resistance from 2Mb to common wheat by inducing CS-Ae. biuncialis homoeologous recombination. Our study also provides useful candidates for further isolation and cloning of powdery mildew resistance gene(s) from Ae. biuncialis chromosome 2Mb.
Project description:Understanding the genetic diversity of Aegilops biuncialis, a valuable source of agronomical useful genes, may significantly facilitate the introgression breeding of wheat. The genetic diversity and population structure of 86 Ae. biuncialis genotypes were investigated by 32700 DArT markers with the simultaneous application of three statistical methods- neighbor-joining clustering, Principal Coordinate Analysis, and the Bayesian approach to classification. The collection of Ae. biuncialis accessions was divided into five groups that correlated well with their eco-geographic habitat: A (North Africa), B (mainly from Balkans), C (Kosovo and Near East), D (Turkey, Crimea, and Peloponnese), and E (Azerbaijan and the Levant region). The diversity between the Ae. biuncialis accessions for a phenological trait (heading time), which is of decisive importance in the adaptation of plants to different eco-geographical environments, was studied over 3 years. A comparison of the intraspecific variation in the heading time trait by means of analysis of variance and principal component analysis revealed four phenotypic categories showing association with the genetic structure and geographic distribution, except for minor differences. The detailed exploration of genetic and phenologic divergence provides an insight into the adaptation capacity of Ae. biuncialis, identifying promising genotypes that could be utilized for wheat improvement.
Project description:<i>Aegilops sharonensis</i>, a wild relative of wheat, harbors diverse disease and insect resistance genes, making it a potentially excellent gene source for wheat improvement. In this study, we characterized and evaluated six wheat-<i>A. sharonensis</i> derivatives, which included three disomic additions, one disomic substitution + monotelosomic addition and two disomic substitution + disomic additions. A total of 51 PLUG markers were developed and used to allocate the <i>A. sharonensis</i> chromosomes in each of the six derivatives to <i>Triticeae</i> homoeologous groups. A set of cytogenetic markers specific for <i>A. sharonensis</i> chromosomes was established based on FISH using oligonucleotides as probes. Molecular cytogenetic marker analysis confirmed that these lines were a CS-<i>A</i>. <i>sharonensis</i> 2S<sup>sh</sup> disomic addition, a 4S<sup>sh</sup> disomic addition, a 4S<sup>sh</sup> (4D) substitution + 5S<sup>sh</sup>L monotelosomic addition, a 6S<sup>sh</sup> disomic addition, a 4S<sup>sh</sup> (4D) substitution + 6S<sup>sh</sup> disomic addition and a 4S<sup>sh</sup> (4D) substitution + 7S<sup>sh</sup> disomic addition line, respectively. Disease resistance investigations showed that chromosome 7S<sup>sh</sup> of <i>A</i>. <i>sharonensis</i> might harbor a new powdery mildew resistance gene, and therefore it has potential for use as resistance source for wheat breeding.
Project description:Diploid Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata are important wild gene sources for wheat. With the aim of assisting in alien gene transfer, this study provides gene-based conserved orthologous set (COS) markers for the U and M genome chromosomes. Out of the 140 markers tested on a series of wheat-Aegilops chromosome introgression lines and flow-sorted subgenomic chromosome fractions, 100 were assigned to Aegilops chromosomes and six and seven duplications were identified in the U and M genomes, respectively. The marker-specific EST sequences were BLAST-ed to Brachypodium and rice genomic sequences to investigate macrosyntenic relationships between the U and M genomes of Aegilops, wheat and the model species. Five syntenic regions of Brachypodium identified genome rearrangements differentiating the U genome from the M genome and from the D genome of wheat. All of them seem to have evolved at the diploid level and to have been modified differentially in the polyploid species Ae. biuncialis and Ae. geniculata. A certain level of wheat-Aegilops homology was detected for group 1, 2, 3 and 5 chromosomes, while a clearly rearranged structure was showed for the group 4, 6 and 7 Aegilops chromosomes relative to wheat. The conserved orthologous set markers assigned to Aegilops chromosomes promise to accelerate gene introgression by facilitating the identification of alien chromatin. The syntenic relationships between the Aegilops species, wheat and model species will facilitate the targeted development of new markers specific for U and M genomic regions and will contribute to the understanding of molecular processes related to allopolyploidization.
Project description:Creation of wheat-alien disomic addition lines and localization of desirable genes on alien chromosomes are important for utilization of these genes in genetic improvement of common wheat. In this study, wheat-Agropyron cristatum derivative line 5113 was characterized by genomic in situ hybridization (GISH) and specific-locus amplified fragment sequencing (SLAF-seq), and was demonstrated to be a novel wheat-A. cristatum disomic 6P addition line. Compared with its parent Fukuhokomugi (Fukuho), 5113 displayed multiple elite agronomic traits, including higher uppermost internode/plant height ratio, larger flag leaf, longer spike length, elevated grain number per spike and spikelet number per spike, more kernel number in the middle spikelet, more fertile tiller number per plant, and enhanced resistance to powdery mildew and leaf rust. Genes conferring these elite traits were localized on the A. cristatum 6P chromosome by using SLAF-seq markers and biparental populations (F1, BC1F1 and BC1F2 populations) produced from the crosses between Fukuho and 5113. Taken together, chromosomal localization of these desirable genes will facilitate transferring of high-yield and high-resistance genes from A. cristatum into common wheat, and serve as the foundation for the utilization of 5113 in wheat breeding.
Project description:BACKGROUND AND AIMS: Repetitive DNA sequences are thought to be involved in the formation of chromosomal rearrangements. The aim of this study was to analyse the distribution of microsatellite clusters in Aegilops biuncialis and Aegilops geniculata, and its relationship with the intergenomic translocations in these allotetraploid species, wild genetic resources for wheat improvement. METHODS: The chromosomal localization of (ACG)(n) and (GAA)(n) microsatellite sequences in Ae. biuncialis and Ae. geniculata and in their diploid progenitors Aegilops comosa and Aegilops umbellulata was investigated by sequential in situ hybridization with simple sequence repeat (SSR) probes and repeated DNA probes (pSc119·2, Afa family and pTa71) and by dual-colour genomic in situ hybridization (GISH). Thirty-two Ae. biuncialis and 19 Ae. geniculata accessions were screened by GISH for intergenomic translocations, which were further characterized by fluorescence in situ hybridization and GISH. KEY RESULTS: Single pericentromeric (ACG)(n) signals were localized on most U and on some M genome chromosomes, whereas strong pericentromeric and several intercalary and telomeric (GAA)(n) sites were observed on the Aegilops chromosomes. Three Ae. biuncialis accessions carried 7U(b)-7M(b) reciprocal translocations and one had a 7U(b)-1M(b) rearrangement, while two Ae. geniculata accessions carried 7U(g)-1M(g) or 5U(g)-5M(g) translocations. Conspicuous (ACG)(n) and/or (GAA)(n) clusters were located near the translocation breakpoints in eight of the ten translocated chromosomes analysed, SSR bands and breakpoints being statistically located at the same chromosomal site in six of them. CONCLUSIONS: Intergenomic translocation breakpoints are frequently mapped to SSR-rich chromosomal regions in the allopolyploid species examined, suggesting that microsatellite repeated DNA sequences might facilitate the formation of those chromosomal rearrangements. The (ACG)(n) and (GAA)(n) SSR motifs serve as additional chromosome markers for the karyotypic analysis of UM genome Aegilops species.
Project description:<i>Aegilops tauschii</i>, 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 <i>Ae. tauschii</i> into durum wheat, however, requires cytogenetic manipulation to induce homoeologous chromosome pairing to promote recombination. For this purpose, the introgression of <i>Ae. tauschii</i> into durum wheat was performed through a bridge cross of the wild species to the Langdon 5D(5B) disomic substitution line that lacks the <i>Ph1</i> locus present on chromosome 5B, followed by a cross of the F<sub>1</sub> 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 <i>Ae. tauschii</i>. Homozygous introgression lines were confirmed using genomic and fluorescence <i>in situ</i> 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 <i>Ae. tauschii</i> 5D segments into durum wheat.
Project description:Thinopyrum ponticum (2n = 10x = 70), a member of the tertiary gene pool of wheat (Triticum aestivum L.), harbors many biotic and abiotic stress resistance genes. CH10A5, a novel disomic substitution line from a cross of T. aestivum cv. 7182 and Th. ponticum, was characterized by cytogenetic identification, in situ hybridization, molecular marker analysis, and morphological investigation of agronomic traits and disease resistance. Cytological observations showed that CH10A5 contained 42 chromosomes and formed 21 bivalents at meiotic metaphase I. Genome in situ hybridization (GISH) analysis indicated that two of its chromosomes came from the Js genome of Th. ponticum, and wheat 15K array mapping and fluorescence in situ hybridization (FISH) revealed that chromosome 1D was absent from CH10A5. Polymorphic analysis of molecular markers indicated that the pair of alien chromosomes belonged to homoeologous group one, designated as 1Js. Thus, CH10A5 was a wheat-Th. ponticum 1Js (1D) disomic substitution line. Field disease resistance trials demonstrated that the introduced Th. ponticum chromosome 1Js was probably responsible for resistance to both stripe rust and powdery mildew at the adult stage. Based on specific-locus amplified fragment sequencing (SLAF-seq), 507 STS molecular markers were developed to distinguish chromosome 1Js genetic material from that of wheat. Of these, 49 STS markers could be used to specifically identify the genetic material of Th. ponticum. CH10A5 will increase the resistance gene diversity of wheat breeding materials, and the markers developed here will permit further tracing of heterosomal chromosome fragments in the future.
Project description: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.
Project description:Cereal grain fiber is an important health-promoting component in the human diet. One option to improve dietary fiber content and composition in wheat is to introduce genes from its wild relatives Aegilops biuncialis and Aegilops geniculata. This study showed that the addition of chromosomes 2Ug, 4Ug, 5Ug, 7Ug, 2Mg, 5Mg, and 7Mg of Ae. geniculata and 3Ub, 2Mb, 3Mb, and 7Mb of Ae. biuncialis into bread wheat increased the seed protein content. Chromosomes 1Ug and 1Mg increased the proportion of polymeric glutenin proteins, while the addition of chromosomes 1Ub and 6Ub led to its decrease. Both Aegilops species had higher proportions of ?-glucan compared to arabinoxylan (AX) than wheat lines, and elevated ?-glucan content was also observed in wheat chromosome addition lines 5U, 7U, and 7M. The AX content in wheat was increased by the addition of chromosomes 5Ug, 7Ug, and 1Ub while water-soluble AX was increased by the addition of chromosomes 5U, 5M, and 7M, and to a lesser extent by chromosomes 3, 4, 6Ug, and 2Mb. Chromosomes 5Ug and 7Mb also affected the structure of wheat AX, as shown by the pattern of oligosaccharides released by digestion with endoxylanase. These results will help to map genomic regions responsible for edible fiber content in Aegilops and will contribute to the efficient transfer of wild alleles in introgression breeding programs to obtain wheat varieties with improved health benefits. Key Message: Addition of Aegilops U- and M-genome chromosomes 5 and 7 improves seed protein and fiber content and composition in wheat.