Periodic Reporting for period 1 - AEGILWHEAT (Widening gene pool of bread wheat by hybridization with Aegilops biuncialis supported by advanced genetic and chromosome genomic approaches)
Periodo di rendicontazione: 2017-09-01 al 2019-08-31
Feeding the 9 billion human population in 2050 will require high yielding, stress tolerant wheat cultivars with good nutritional quality to produce enough food by sustainable and cost effective means with reduced chemical load of the environment.
Breeding of improved wheat varieties could be accelerated by the use of genes which are absent in extant gene pool of hexaploid wheat. The wheat wild relative Aegilops species represent a rich source of genes for important traits including grain yield, quality and tolerance to abiotic and biotic stress, which can be utilized by interspecific hybridization. To date, the application of wild genetic diversity in breeding programs has been hampered by low throughput of methods used to select introgression lines and the lack of knowledge on the genomes of wild relatives.
The objectives of multidisciplinary AEGILWHEAT project were to (1) isolate individual chromosomes 1Ub, 3Ub, 3Mb and 7Mb from Aegilops biuncialis and (2) identify their gene content and produce polymorphic markers (SNPs, INDELs) by next-generation sequencing and bioinformatic analyses. The genes and new markers will be integrated into (3) a new high resolution genetic linkage map of Ae. biuncialis. The marker-assisted selection system will be used for the (4) high throughput selection of wheat-Ae. biuncialis introgression lines.
Breeding of improved wheat varieties could be accelerated by the use of genes which are absent in extant gene pool of hexaploid wheat. The wheat wild relative Aegilops species represent a rich source of genes for important traits including grain yield, quality and tolerance to abiotic and biotic stress, which can be utilized by interspecific hybridization. To date, the application of wild genetic diversity in breeding programs has been hampered by low throughput of methods used to select introgression lines and the lack of knowledge on the genomes of wild relatives.
The objectives of multidisciplinary AEGILWHEAT project were to (1) isolate individual chromosomes 1Ub, 3Ub, 3Mb and 7Mb from Aegilops biuncialis and (2) identify their gene content and produce polymorphic markers (SNPs, INDELs) by next-generation sequencing and bioinformatic analyses. The genes and new markers will be integrated into (3) a new high resolution genetic linkage map of Ae. biuncialis. The marker-assisted selection system will be used for the (4) high throughput selection of wheat-Ae. biuncialis introgression lines.
We started the project with the isolation of Ae. biuncialis chromosomes by flow cytometric sorting. The bivariate flow karyotyping based on GAA-FITC signals on the DAPI stained chromosomes was able to resolve two populations specific for single chromosomes 1Ub and 7Mb that could be sorted in high purity (92-98%). The remaining chromosomes could be sorted only in groups of 3-4 from Ae. biuncialis, so the chromosomes 3Ub and 3Mb and also 2Mb and 6Ub were sorted from wheat-Ae. biuncialis chromosome addition lines in purity of 90% - 96%. We were also able to sort the whole set of U- and M-genome chromosomes from the diploid progenitors, Ae. umbellulata (UU) and Ae. comosa (MM) at purities 88-99% and 72-89%, respectively.
Thanks to the successful chromosome sorting, the chromosomes 1Ub, 3Ub, 3Mb and 7Mb of Ae. biuncialis could be sequenced. Moreover, we also sequenced the whole U and M genomes of diploid Aegilops by chromosomes (1U-7U and 1M-7M). By the end of the project, we determined the gene content of the chromosomes 1Ub and 1U-7U. The comparative analysis highlighted several evolutionary rearrangements in the U genome relative to wheat.
We developed gene-based markers using sequence similarity approach. The alignment of wheat genic EST sequences to chromosome contigs of Ae. umbellulata resulted in 445 hits with >5bp INDELs. In the frame of the project, 122 INDEL-based markers were tested by PCR on wheat, Ae. biuncialis and Ae. umbellulata, 51 were polymorphic and 21 markers were assigned to single chromosomes of Aegilops (1U: 6, 2U:1, 4U:4, 5U:3, 6U:3, 7U: 1, 1M: 1, 5M:2). These markers will be suitable for the marker assisted selection of new wheat-Aegilops introgression lines.
In order to produce genetic map, we applied DArTseq, a genotyping-by-sequencing technology to determine the allel composition of 5893 and 22,180 quality filtered SNP-DArT and Silico-DArT markers, respectively, on the Ae. biuncialis F2 population. The present version of the genetic map contains 17 linkage groups (LGs), where the chromosomes 5U, 6U and 1M represented by two and two LGs, respectively. The total length of the map is 2758.08 cM, containing 1002 individual loci and 13,811 markers. The average distance between two loci is 2.91 cM and the biggest gap is 23.94 cM.
The genetic variability of wheat has also been improved by the transfer of new Ae. biuncialis chromosomes and chromosome segments. We detected several Aegilops chromosomes in the wheat-Ae. biuncialis hybrid progenies by molecular cytogenetic methods suitable to visualize (GISH) and identify (FISH) the alien chromosomes. We obtained new wheat-Ae. biuncialis translocations, which was an important goal of the project. The most important results of the project were the wheat-Ae. biuncialis translocations in disomic form in some plants. The terminal translocations T1DL.1DS-U T2DS.2DL-U T4DL.4DS-M T5DS.5DL-M will be characterized for agronomically important traits and transferred into advanced wheat cultivars to use in the breeding programs. We also detected wheat-Aegilops chromosome rearrangements where a wheat chromosome segment translocated to an Aegilops chromosome (T6US.6UL-6BL T2MS.2ML-2D). These translocations will be applied to induce further rearrangements between the wheat and Aegilops chromosomes.
Thanks to the successful chromosome sorting, the chromosomes 1Ub, 3Ub, 3Mb and 7Mb of Ae. biuncialis could be sequenced. Moreover, we also sequenced the whole U and M genomes of diploid Aegilops by chromosomes (1U-7U and 1M-7M). By the end of the project, we determined the gene content of the chromosomes 1Ub and 1U-7U. The comparative analysis highlighted several evolutionary rearrangements in the U genome relative to wheat.
We developed gene-based markers using sequence similarity approach. The alignment of wheat genic EST sequences to chromosome contigs of Ae. umbellulata resulted in 445 hits with >5bp INDELs. In the frame of the project, 122 INDEL-based markers were tested by PCR on wheat, Ae. biuncialis and Ae. umbellulata, 51 were polymorphic and 21 markers were assigned to single chromosomes of Aegilops (1U: 6, 2U:1, 4U:4, 5U:3, 6U:3, 7U: 1, 1M: 1, 5M:2). These markers will be suitable for the marker assisted selection of new wheat-Aegilops introgression lines.
In order to produce genetic map, we applied DArTseq, a genotyping-by-sequencing technology to determine the allel composition of 5893 and 22,180 quality filtered SNP-DArT and Silico-DArT markers, respectively, on the Ae. biuncialis F2 population. The present version of the genetic map contains 17 linkage groups (LGs), where the chromosomes 5U, 6U and 1M represented by two and two LGs, respectively. The total length of the map is 2758.08 cM, containing 1002 individual loci and 13,811 markers. The average distance between two loci is 2.91 cM and the biggest gap is 23.94 cM.
The genetic variability of wheat has also been improved by the transfer of new Ae. biuncialis chromosomes and chromosome segments. We detected several Aegilops chromosomes in the wheat-Ae. biuncialis hybrid progenies by molecular cytogenetic methods suitable to visualize (GISH) and identify (FISH) the alien chromosomes. We obtained new wheat-Ae. biuncialis translocations, which was an important goal of the project. The most important results of the project were the wheat-Ae. biuncialis translocations in disomic form in some plants. The terminal translocations T1DL.1DS-U T2DS.2DL-U T4DL.4DS-M T5DS.5DL-M will be characterized for agronomically important traits and transferred into advanced wheat cultivars to use in the breeding programs. We also detected wheat-Aegilops chromosome rearrangements where a wheat chromosome segment translocated to an Aegilops chromosome (T6US.6UL-6BL T2MS.2ML-2D). These translocations will be applied to induce further rearrangements between the wheat and Aegilops chromosomes.
The results of the project will serve as a foundation for the exploitation of wild genetic diversity in the production of stress tolerant wheat cultivars. Our innovative and advanced flow cytometric study showed that the U and M genomes of wild Aegilops species can be dissected into chromosomes which can be used for various genomic studies including next generation sequencing. We are sure that our flow cytometric method can be applicable to a wide range of gene source species in the taxa of Triticum and Aegilops. The first chromosome-based sequence assembly of U and M genomes and the genetic map of Ae. biuncialis will facilitate the structural and functional genomic studies, like map-based cloning of agronomically important genes. The genetic map and the produced markers will make the transfer of Aegilops chromosome segments to wheat more efficient. The project already produced six wheat-Ae. biuncialis translocation lines. As the Aegilops crossing partner line was resistant to rust diseases, the high priority target will be to identify the chromosome containing the resistance gene and produce wheat-Aegilops translocation conferring leaf rust resistance to wheat.
In this context the disease resistant wheat cultivars with Aegilops chromosome segments will promote the environment friendly food production by decreasing the use of chemicals (fungicides). The resistant cultivars will also promote the production of healthy foods and the protection of (agricultural) environment.
In summary, the genomic knowledge on wild genetic resources provided by the AEGILWHEAT project and its expected results, the new wheat cultivars will be important tools for the policy makers to cope with the actual environmental and ecological risks.
In this context the disease resistant wheat cultivars with Aegilops chromosome segments will promote the environment friendly food production by decreasing the use of chemicals (fungicides). The resistant cultivars will also promote the production of healthy foods and the protection of (agricultural) environment.
In summary, the genomic knowledge on wild genetic resources provided by the AEGILWHEAT project and its expected results, the new wheat cultivars will be important tools for the policy makers to cope with the actual environmental and ecological risks.