Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii, Ae. triuncialis and Ae. cylindrica, and their molecular organization.
ABSTRACT: Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U(t)U(t)C(t)C(t)) and Ae. cylindrica (D(c)D(c)C(c)C(c)) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting.The flow karyotypes obtained after the analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA.FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C(t), T6U(t)S.6U(t)L-5C(t)L, 1C(c) and 5D(c) could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2-5. This identified a partial wheat-C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C-2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected.The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.
Project description:Aegilops caudata is an important gene source for wheat breeding. Intensive evaluation of its utilization value is an essential first step prior to its application in breeding. In this research, the agronomical and quality traits of Triticum aestivum-Ae. caudata additions B-G (homoeologous groups not identified) were analyzed and evaluated. Disease resistance tests showed that chromosome D of Ae. caudata might possess leaf rust resistance, and chromosome E might carry stem rust and powdery mildew resistance genes. Investigations into agronomical traits suggested that the introduction of the Ae. caudata chromosome in addition line F could reduce plant height. Grain quality tests showed that the introduction of chromosomes E or F into wheat could increase its protein and wet gluten content. Therefore, wheat-Ae. caudata additions D-F are all potentially useful candidates for chromosome engineering activities to create useful wheat-alien chromosome introgressions. A total of 55 EST-based molecular markers were developed and then used to identify the chromosome homoeologous group of each of the Ae. caudata B-G chromosomes. Marker analysis indicated that the Ae. caudata chromosomes in addition lines B to G were structurally altered, therefore, a large population combined with intensive screening pressure should be taken into consideration when inducing and screening for wheat-Ae. caudata compensating translocations. Marker data also indicated that the Ae. caudata chromosomes in addition lines C-F were 5C, 6C, 7C, and 3C, respectively, while the homoeologous group of chromosomes B and G of Ae. caudata are as yet undetermined and need further research.
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:Gene flow between domesticated species and their wild relatives is receiving growing attention. This study addressed introgression between wheat and natural populations of its wild relatives (Aegilops species). The sampling included 472 individuals, collected from 32 Mediterranean populations of three widespread Aegilops species (Aegilops geniculata, Ae. neglecta and Ae. triuncialis) and compared wheat field borders to areas isolated from agriculture. Individuals were characterized with amplified fragment length polymorphism fingerprinting, analysed through two computational approaches (i.e. Bayesian estimations of admixture and fuzzy clustering), and sequences marking wheat-specific insertions of transposable elements. With this combined approach, we detected substantial gene flow between wheat and Aegilops species. Specifically, Ae. neglecta and Ae. triuncialis showed significantly more admixed individuals close to wheat fields than in locations isolated from agriculture. In contrast, little evidence of gene flow was found in Ae. geniculata. Our results indicated that reproductive barriers have been regularly bypassed during the long history of sympatry between wheat and Aegilops.
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.
Project description:The bread wheat (Triticum aestivum L.) genotype "Chinese Spring" ("CS") is the reference base in wheat genetics and genomics. Pericentric rearrangements in this genotype were systematically assessed by analyzing homoeoloci for a set of nonredundant genes from Brachypodium distachyon, Triticum urartu, and Aegilops tauschii in the CS chromosome shotgun sequence obtained from individual chromosome arms flow-sorted from CS aneuploid lines. Based on patterns of their homoeologous arm locations, 551 genes indicated the presence of pericentric inversions in at least 10 of the 21 chromosomes. Available data from deletion bin-mapped expressed sequence tags and genetic mapping in wheat indicated that all inversions had breakpoints in the low-recombinant gene-poor pericentromeric regions. The large number of putative intrachromosomal rearrangements suggests the presence of extensive structural differences among the three subgenomes, at least some of which likely occurred during the production of the aneuploid lines of this hexaploid wheat genotype. These differences could have significant implications in wheat genome research where comparative approaches are used such as in ordering and orientating sequence contigs and in gene cloning.
Project description:The 'breaker' element ( GcB ) of the gametocidal locus derived from Aegilops sharonensis has been mapped to a region proximal to a block of sub-telomeric heterochromatin on chromosome 4S (sh) L. The production of alien chromosome addition lines allows the transfer of useful genetic variation into elite wheat varieties from related wild species. However, some wild relatives of wheat, particularly those within the Sitopsis section of the genus Aegilops, possess chromosomes that are transmitted preferentially to the offspring when addition lines are generated. Species within the Sitopsis group possess the S genome, and among these species, Aegilops sharonensis (2n = 14, S(sh)S(sh)) carries the S(sh) genome which is closely related to the D genome of hexaploid wheat. Some S genome chromosomes carry gametocidal loci, which induce severe chromosome breakage in gametes lacking the gametocidal chromosome, and hence, result in gamete abortion. The preferential transmission of gametocidal loci could be exploited in wheat breeding, because linking gametocidal loci with important agronomic traits in elite wheat varieties would ensure retention of these traits through successive generations. In this study, we have mapped the breaker element of the gametocidal locus derived from Ae. sharonensis to the region immediately proximal to a block of sub-telomeric heterochromatin on the long arm of chromosome 4S(sh).
Project description:Wild relatives of wheat, such as Aegilops spp. are potential sources of genes conferring tolerance to drought stress. As drought stress affects seed composition, the main goal of the present study was to determine the effects of drought stress on the content and composition of the grain storage protein (gliadin (Gli), glutenin (Glu), unextractable polymeric proteins (UPP%) and dietary fiber (arabinoxylan, ?-glucan) components of hexaploid bread wheat (T. aestivum) lines containing added chromosomes from Ae. biuncialis or Ae. geniculata. Both Aegilops parents have higher contents of protein and ?-glucan and higher proportions of water-soluble arabinoxylans (determined as pentosans) than wheat when grown under both well-watered and drought stress conditions. In general, drought stress resulted in increased contents of protein and total pentosans in the addition lines, while the ?-glucan content decreased in many of the addition lines. The differences found between the wheat/Aegilops addition lines and wheat parents under well-watered conditions were also manifested under drought stress conditions: Namely, elevated ?-glucan content was found in addition lines containing chromosomes 5Ug, 7Ug and 7Mb, while chromosomes 1Ub and 1Mg affected the proportion of polymeric proteins (determined as Glu/Gli and UPP%, respectively) under both well-watered and drought stress conditions. Furthermore, the addition of chromosome 6Mg decreased the WE-pentosan content under both conditions. The grain composition of the Aegilops accessions was more stable under drought stress than that of wheat, and wheat lines with the added Aegilops chromosomes 2Mg and 5Mg also had more stable grain protein and pentosan contents. The negative effects of drought stress on both the physical and compositional properties of wheat were also reduced by the addition of these. These results suggest that the stability of the grain composition could be improved under drought stress conditions by the intraspecific hybridization of wheat with its wild relatives.
Project description:Chromosome translocations can be detected by cytogenetic analysis, but it is hard to characterize the breakpoints at the sequence level. Chromosome sorting by flow cytometry produces flow karyotypes that enable the isolation of abnormal chromosomes and the generation of chromosome-specific DNA. In this study, a derivative chromosome t(9; 14) and its homologous normal chromosomes 9 and 14 from the Ishikawa 3-H-12 cell line were sorted to collect homologue-specific samples. Chromosome sequencing identified the breakpoint junction in the der(9) at 9p24.3 and 14q13.1 and uncovered the formation of a fusion gene, WASH1-NPAS3. Amplicon sequencing targeted for neighbouring genes at the fusion breakpoint revealed that the variant frequencies correlate with the allelic copy number. Sequencing of sorted chromosomes permits the assignment of allelic variants and can lead to the characterization of abnormal chromosomes. We show that allele-specific chromosome sequencing of homologues is a robust technique for distinguishing alleles and this provides an efficient approach for the comprehensive analysis of genomic changes.
Project description:A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement. To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6 x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1 U-6 U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3 L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.
Project description:This study characterized and evaluated a set of wheat-Aegilops comosa introgression lines, including six additions and one substitution. A total of 47 PLUG markers and a set of cytogenetic markers specific for Ae. comosa chromosomes were established after screening 526 PLUG primer pairs and performing FISH using oligonucleotides as probes. Marker analysis confirmed that these lines were wheat-Ae. comosa 2M-7M addition lines and a 6M(6A) substitution line. The molecular and cytogenetic markers developed herein could be used to trace Ae. comosa chromatin in wheat background. In order to evaluate the breeding value of the material, disease resistance tests and agronomical trait investigations were carried out on these alien chromosome introgression lines. Disease resistance tests showed that chromosomes 2M and 7M of Ae. comosa might harbor new stripe rust and powdery mildew resistance genes, respectively, therefore, they could be used as resistance sources for wheat breeding. Investigations into agronomical traits showed that all chromosomes 2M to 7M had detrimental effects on the agronomic performance of wheat, therefore, the selection of plants with relatively negative effects should be avoided when inducing wheat-A. comosa chromosome translocations using chromosome engineering procedures.