Novel molecular marker-assisted strategy for production of wheat-Leymus mollis chromosome addition lines.
ABSTRACT: Developing wheat-alien chromosome introgression lines to improve bread wheat's resistance to stresses, such as drought, salinity stress and diseases, requires reliable markers to identify and characterize the alien chromatins. Leymus mollis is a wild relative of bread wheat resistant to salinity and economically important diseases of wheat, but its genome sequence and cytological markers are not available. We devised a molecular marker-assisted strategy for L. mollis chromosome identification and applied it to produce 10 wheat-L. mollis chromosome addition lines. Using 47?L. racemosus genome polymorphic PCR markers and DArTseq genotyping, we distinguished the L. mollis chromosomes and differentiated disomic and monosomic lines by progeny test. DArTseq genotyping generated 14,530?L. mollis SNP markers and the chromosome-specific SNP markers were used to determine the homoeologous groups of L. mollis chromosomes in the addition lines. To validate the marker-based results, genomic in situ hybridization was applied to confirm the presence and cytological status of L. mollis chromosomes in the lines. This study demonstrates that adequate molecular markers allow the production and characterization of wheat-alien addition lines without in situ hybridization, which saves considerable time and effort.
Project description:BACKGROUND:The tertiary gene pool of bread wheat, to which Leymus racemosus belongs, has remained underutilized due to the current limited genomic resources of the species that constitute it. Continuous enrichment of public databases with useful information regarding these species is, therefore, needed to provide insights on their genome structures and aid successful utilization of their genes to develop improved wheat cultivars for effective management of environmental stresses. RESULTS:We generated de novo DNA and mRNA sequence information of L. racemosus and developed 110 polymorphic PCR-based markers from the data, and to complement the PCR markers, DArT-seq genotyping was applied to develop additional 9990 SNP markers. Approximately 52% of all the markers enabled us to clearly genotype 22 wheat-L. racemosus chromosome introgression lines, and L. racemosus chromosome-specific markers were highly efficient in detailed characterization of the translocation and recombination lines analyzed. A further analysis revealed remarkable transferability of the PCR markers to three other important Triticeae perennial species: L. mollis, Psathyrostachys huashanica and Elymus ciliaris, indicating their suitability for characterizing wheat-alien chromosome introgressions carrying chromosomes of these genomes. CONCLUSION:The efficiency of the markers in characterizing wheat-L. racemosus chromosome introgression lines proves their reliability, and their high transferability further broadens their scope of application. This is the first report on sequencing and development of markers from L. racemosus genome and the application of DArT-seq to develop markers from a perennial wild relative of wheat, marking a paradigm shift from the seeming concentration of the technology on cultivated species. Integration of these markers with appropriate cytogenetic methods would accelerate development and characterization of wheat-alien chromosome introgression lines.
Project description:<h4>Background</h4>Triticum kiharae (A<sup>t</sup>A<sup>t</sup>GGDD, 2n?=?42) is of interest for the improvement of bread wheat as a source of high grain protein and gluten content, as well as resistance to many diseases. The use of T. kiharae for the improvement of T. aestivum L. is complicated by the fact that the homology degree of their genomes is low and this leads to an unbalanced set of chromosomes in the gametes of its first generations and the elimination of some genotypes. The aim of this study was to analyze the nature of alien introgressions and their effect on the cytological stability of hybrids obtained from crossing of bread wheat varieties with T. kiharae.<h4>Results</h4>Using C-banding, the presence of entire chromosomes of T. kiharae in the karyotypes of hybrid lines (intergenomic substitution 2G/2B), chromosome arms (centric translocation ?2A<sup>t</sup>S:2AL) and large inserts in the form of terminal translocations involving chromosomes of 1st, 3rd and 5th homoeologous groups of B- and G-genomes were found. Molecular markers revealed short introgression of T. kiharae into the genome of common wheat varieties. The highest introgression frequency was shown for 1A, 1B, 2A, 5B, and 6A chromosomes, while no foreign chromatin was detected in 4A and 4B chromosomes. A high level of cytological stability (a meiotic index of 88.18-93.0%) was noted for the majority of introgression lines. An exception was found for the lines containing the structural reorganization of chromosome 5B, affecting the main genes of chromosome synapsis in terms of their functioning.<h4>Conclusions</h4>During the stabilization of hybrid karyotypes, the introgression of genetic material from T. kiharae into the genome of T. aestivum occurs in the form of short fragments detectable only by molecular markers and in the form of whole chromosomes (intergenomic substitution) and their large fragments (centric and terminal translocations). The level of cytological stability achieved in F<sub>10</sub> by the majority of introgression lines ensures the formation of functional gametes sufficient for the successful reproduction of the obtained hybrids.
Project description: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.
Project description:KEY MESSAGE:Using COS markers, the study reveals homeologous relationships between tetraploid Agropyron cristatum and bread wheat to support alien introgression breeding of wheat. Crested wheatgrass (Agropyron cristatum L. Gaertn.) is a wild relative of wheat that possesses many genes that are potentially useful in wheat improvement. The species comprises a complex of diploid, tetraploid and hexaploid forms. In this study, wheat-A. cristatum chromosome, telosome and translocation lines were used to characterize syntenic relationships between tetraploid A. cristatum and bread wheat. Prior to mapping COS markers, the cytogenetic stock lines were characterized for fertility and by FISH and GISH for karyotype stability. Out of 328 COS markers selected for the study, 279 consistently amplified products in tetraploid A. cristatum, and, out of these, 139 were polymorphic between tetraploid crested wheatgrass and wheat. Sixty-nine markers were found to be suitable for the detection of tetraploid A. cristatum chromosomes 1P-6P in wheat, ranging from 6 to 17 markers per chromosome. BLASTn of the source ESTs resulted in significant hits for 67 markers on the wheat pseudomolecules. Generally, COS markers of the same homeologous group were detected on similar arms in both Agropyron and wheat. However, some intragenomic duplications and chromosome rearrangements were detected in tetraploid A. cristatum. These results provide new insights into the structure and evolution of the tetraploid A. cristatum genome and will facilitate the exploitation of the wild species for introgression breeding of bread wheat.
Project description:Agropyron cristatum (L.) Gaertn. (2n = 28, PPPP), a wild relative of common wheat, possesses many potentially valuable traits that can be transferred to common wheat through breeding programs. The wheat-A. cristatum disomic addition and translocation lines can be used as bridge materials to introduce alien chromosomal segments to wheat. Wheat-A. cristatum 2P disomic addition line II-9-3 was highly resistant to powdery mildew and leaf rust, which was reported in our previous study. However, some translocation lines induced from II-9-3 have not been reported. In this study, some translocation lines were induced from II-9-3 by 60Co-? irradiation and gametocidal chromosome 2C and then identified by cytological methods. Forty-nine wheat-A. cristatum translocation lines were obtained and various translcoation types were identified by GISH (genomic in situ hybridization), such as whole-arm, segmental and intercalary translocations. Dual-color FISH (fluorescent in situ hybridization) was applied to identify the wheat chromosomes involved in the translocations, and the results showed that A. cristatum 2P chromosome segments were translocated to the different wheat chromosomes, including 1A, 2A, 3A, 4A, 5A, 6A, 7A, 3B, 5B, 7B, 1D, 4D and 6D. Many different types of wheat-A. cristatum alien translocation lines would be valuable for not only identifying and cloning A. cristatum 2P-related genes and understanding the genetics and breeding effects of the translocation between A. cristatum chromosome 2P and wheat chromosomes, but also providing new germplasm resources for the wheat genetic improvement.
Project description: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:BACKGROUND:Dasypyrum villosum is an important wild species of wheat (Triticum aestivum L.) and harbors many desirable genes that can be used to improve various traits of wheat. Compared with other D. villosum accessions, D. villosum#4 still remains less studied. In particular, chromosomes of D. villosum#4 except 6V#4 have not been introduced into wheat by addition or substitution and translocation, which is an essential step to identify and apply the alien desired genes. RNA-seq technology can generate large amounts of transcriptome sequences and accelerate the development of chromosome-specific molecular markers and assisted selection of alien chromosome line. RESULTS:We obtained the transcriptome of D. villosum#4 via a high-throughput sequencing technique, and then developed 76 markers specific to each chromosome arm of D. villosum#4 based on the bioinformatic analysis of the transcriptome data. The D. villosum#4 sequences containing the specific DNA markers were expected to be involved in different genes, among which most had functions in metabolic processes. Consequently, we mapped these newly developed molecular markers to the homologous chromosome of barley and obtained the chromosome localization of these markers on barley genome. Then we analyzed the collinearity of these markers among D. villosum, wheat, and barley. In succession, we identified six types of T. aestivum-D. villosum#4 alien chromosome lines which had one or more than one D. villosum#4 chromosome in the cross and backcross BC3F5 populations between T. durum-D. villosum#4 amphidiploid TH3 and wheat cv. Wan7107 by employing the selected specific markers, some of which were further confirmed to be translocation or addition lines by genomic in situ hybridization (GISH). CONCLUSION:Seventy-six PCR markers specific to chromosomes of D. villosum#4 based on transcriptome data were developed in the current study and their collinearity among D. villosum, wheat, and barley were carried out. Six types of Triticum aestivum-D. villosum#4 alien chromosome lines were identified by using 12 developed markers and some of which were further confirmed by GISH. These novel T. aestivum-D. villosum#4 chromosome lines have great potential to be used for the introduction of desirable genes from D. villosum#4 into wheat by chromosomal translocation to breed new wheat varieties.
Project description:Meiotic pairing between homoeologous chromosomes in polyploid wheat is inhibited by the Ph1 locus on the long arm of chromosome 5 in the B genome. Aegilops speltoides (genomes SS), the closest relative of the progenitor of the wheat B genome, is polymorphic for genetic suppression of Ph1. Using this polymorphism, two major suppressor loci, Su1-Ph1 and Su2-Ph1, have been mapped in Ae. speltoides. Su1-Ph1 is located in the distal, high-recombination region of the long arm of the Ae. speltoides chromosome 3S. Its location and tight linkage to marker Xpsr1205-3S makes Su1-Ph1 a suitable target for introgression into wheat. Here, Xpsr1205-3S was introgressed into hexaploid bread wheat cv. Chinese Spring (CS) and from there into tetraploid durum wheat cv. Langdon (LDN). Sequential fluorescence in situ hybridization and genomic in situ hybridization showed that an Ae. speltoides segment with Xpsr1205-3S replaced the distal end of the long arm of chromosome 3A. In the CS genetic background, the chromosome induced homoeologous chromosome pairing in interspecific hybrids with Ae. peregrina but not in progenies from crosses involving alien disomic substitution lines. In the LDN genetic background, the chromosome induced homoeologous chromosome pairing in both interspecific hybrids and progenies from crosses involving alien disomic substitution lines. We conclude that the recombined chromosome harbors Su1-Ph1 but its expression requires expression of complementary gene that is present in LDN but absent in CS. We suggest that it is unlikely that Su1-Ph1 and ZIP4-1, a paralog of Ph1 located on wheat chromosomes 3A and 3B and Ae. tauschii chromosome 3D, are equivalent. The utility of Su1-Ph1 for induction of recombination between homoeologous chromosomes in wheat is illustrated.
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: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.