Progress 02/15/16 to 02/14/19
Outputs
Target Audience:The research results from this work have been disseminated by the PD and graduate students at national or international meetings (see above for the details), invited talks and annual departmental seminars. The audiences for these meetings and seminars included fungal geneticists and biologists, plant geneticists and biologists, graduate students, post-doctoral researchers and other professionals. The published peer-reviewed articles can reach a broader audience in the world scientific community. ? Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph.D students, Gayan Kanishka Kariyawasam and Jingwei Guo, in the Department of Plant Pathology, North Dakota State University are working on this project and have been training in fungal genetics and biology and professional development from this project. Two undergraduate student, Joseph Lead majoring in biology and Kristen Almen majoring in agronomy and environmental science at NDSU, received training in molecular genetics and microbiology. Ms. Almen has started a graduate program at NDSU. How have the results been disseminated to communities of interest?The results from this project have been disseminated by the PD and graduate students at international or national meetings, including the 29th Fungal Genetics Conference in Pacific Grove, CA (March 14-18, 2017), the 2017 Dothideomycetes Comparative Genomics workshop in Pacific Grove, CA, and the 2018 American Phytopathological Society North Central Division Meeting in Fargo, ND (June 12-14, 2018). During these meeting, the research results were disseminated by the way of poster or oral presentations as well as published abstracts in meeting program books. The PD attended the NIFA-AFRI project directory meetings in Washington DC twice during this project (June 30-July 1, 2016, and December 11-12, 2017) and reported the research results to other PDs. Graduate students also presented research results at departmental seminars. Two peer-reviewed articles have been published from this project with other two being prepared for publication. ? What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
For objective 1) The heterothallic fungal strain by deleting one of mating type gene was createdin isolates 86-124 (race 2), DW5 (race 5), AR CrossB10 (new race), L13-192L2 (race 4), and by using the heterothallic strains, the fungal populations have been successfully developed from the crosses of 86-124×DW5, AR CrossB10×DW5, AR CrossB10×86-124 with the number of progeny at 118, 164, and 144, respectively. However, the crosses between L13-192 with other isolates did not produce any fertile pseudothecia probably due to genetic distance and incompatibility between race 4 and other races. For objective 2) Because AR CrossB10 produces Ptr ToxC and 86-124 does not, the population AR CrossB10×86-124, which thereafter was referred to AR/86, was used in the genetic mapping of fungal gene(s) responsible for the Ptr ToxC production. The AR/86 population segregates for the Ptr ToxC production as 50 (Ptr ToxC producing):62 (non-Ptr ToxC producing), which fits the 1:1 ratio (χ2=1.28, P=0.25) suggesting a single gene conditioning Ptr ToxC production. However, the segregation for Ptr ToxA production was 46 (producing Ptr ToxA):67 (producing no Ptr ToxC), which slightly deviates from 1:1 (χ2=4.32, P=0.04). Due to the low polymorphism between two parental lines, the restriction-site-associated DNA sequencing method (about 1x coverage) did allow us to develop any polymorphic marker for genetic mapping. Therefore, we sequenced a total of 20 progeny (10 producing Ptr ToxC and 10 producing no Ptr ToxC) as well as the two parental isolates with about 50 x coverage using illumina Hiseq platform. SNPs were mined from the genome sequences for marker development and linkage mapping. The resulting map contains 16 linkage groups spanning 4922.8 cM in genetic distance. Genetic mapping with Ptr ToxC phenotypic data indicate the gene responsible for the production is located in the distal of chromosome 2 and candidate gene is delimited to about a ~200 kb genomic region in the reference genome. To map fungal gene in DW5 that confers necrosis development on 'rusty' durum wheat, we conducted the genotyping and phenotyping of the 86-124×DW5 (86/DW) population. In this population, the segregation ratios of the mating type gene, ToxA, and ToxB were 58:60 (MAT1-1:MAT1-2), 45:73 (present vs absent) and 96:22 (present vs absent), respectively. The 1:1 segregation ratio for the mating type gene indicates the developed population has no problem. The significant deviation of the ToxA gene segregation ratio from 1:1 is not expected, which is intriguing. The segregation ratio for the presence of ToxB is close to 3:1 indicating two independent genetic loci for ToxB. The DW5 probably has six copies of ToxB with five being in one locus and one in the other locus. The locus containing five copies is on the chromosome 11 and the other locus is likely on the chromosome 2. Phenotyping data suggests that ToxB likely confers the necrosis development on rusty. To identify new host and necrotrophic effector interaction, we evaluated a wheat population for reaction to several progeny which have neither ToxA nor ToxB. QTL mapping indicates a wheat gene on the chromosome 7B conditions disease susceptibility to the new NE.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Guo, J., Shi, G., Liu, Z.H. 2018. Characterizing Virulence of the Pyrenophora tritici-repentis Isolates Lacking Both ToxA and ToxB Genes. Pathogens, 7(3):74 https://doi.org/10.3390/pathogens7030074
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2018
Citation:
Kariyawasam, G. MOLECULAR GENETIC CHARACTERIZATION OF PTR TOXC-TSC1 INTERACTION AND COMPARATIVE GENOMICS OF PYRENOPHORA TRITICI-REPENTIS. North Dakota State University, PhD dissertation
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Progress 02/15/17 to 02/14/18
Outputs
Target Audience:The research results from this period work have been disseminated by the PD and graduate students at national or international meetings, including the 29th Fungal Genetics Conferrence in Pacific Grove, CA, March 14-18, 2017 and the NIFA-AFRI Project Directory meeting in Washington DC, December 11-12, 2017. The results were also presented in annual departmental seminars. The audiences for these meetings and seminars included fungal geneticists and biologists, plant geneticists and biologists, graduate students, post-doctoral researchers and other professonials. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph.D students, Gayan Kanishka Kariyawasam and Jingwei Guo, in the Department of Plant Pathology, North Dakota State University are working on this project and have been training in fungal genetics and biology and professional development from this project. One undergraduate student, Kristen Almen majoring in agronomy and environmental science at NDSU received training in molecular genetics and microbiology. How have the results been disseminated to communities of interest?The results from this project have been disseminated by the PD and graduate students at international or national meetings or departmental seminars. What do you plan to do during the next reporting period to accomplish the goals?We will finish the phenotyping and genotyping of the fungal population derived from 86-124×DW5 and the gene for virulence toward 'rusty' or 'Kronos' will be mapped. The fungal progeny without ToxA and ToxB will be tested on several wheat populations for identifying new necrotrophic effector and host sensivity gene interactions.
Impacts
What was accomplished under these goals?
The research work associated with the objective 1 has been basically accomplished, which was the development of several fungal populations. Our work for this peroid was mainly focused on the objective 2. We have completed phenotyping and genotyping of the fungal population derived from AR Crossb10 x 86-124 (referred to as AR/86 population) which segregates for the production of Ptr ToxC. The AR/86 population segregates for the Ptr ToxC production as 50 (Ptr ToxC producing):62 (non-Ptr ToxC producing), which fits the 1:1 ratio (χ2=1.28, P=0.25) suggesting a single gene conditioning Ptr ToxC production. However, the segregation for Ptr ToxA production was 46 (producing Ptr ToxA):67 (producing no Ptr ToxC), which slightly deviates from 1:1 (χ2=4.32, P=0.04). The restriction-site-associated DNA sequencing (RAD-seq) method (about 1x coverage) was first used to genotype the population, but no polymorphic marker was obtained. Subsequently, a total of 20 progeny (10 producing Ptr ToxC and 10 producing no Ptr ToxC) as well as the three parental isolates were sequenced for the whole genome with about 50 x coverage using illumina Hiseq platform. Sequencing analysis showed an extremely low level of SNP (0.3 SNP/kb) between two parental lines, which explains well why our RAD-seq method failed. Genome comparison among the sequenced Ptr ToxC producing and non-Ptr ToxC producing progeny showed a genomic region on chromosome 2 associated with Ptr ToxC production. The SNP markers around that region were developed and genotyped on the whole population leading to the identification of a ~40 kb candidate region on that chromosome. There are ten genes predicted in the candidate region. The second population that we developed was derived from 86-124×DW5, refered to the 86/DW population. 'Rusty', 'Kronos' and some other durum lines are resistant to 86-124, but highly susceptible (developing large necrotic lesions) to DW5. Thus, the 86/DW population is useful for mapping virulence gene in DW5 towards Rusty. For this population, the segregation ratios of mating type gene, ToxA, and ToxB were 58:60 (MAT1-1:MAT1-2), 45:73 (present vs absent) and 96:22 (present vs absent). The deviation of ToxB segregation ratio from 1:1 may be due to the presence of multiple copies of ToxB gene in DW5. The significant deviation of the ToxA gene segregation ratio from 1:1 is not expected, which is intriguing. We obtained fungal progenies that have recombinant genotypes with neither ToxA or ToxB gene. We have genotyped the population with 60 SSR markers, the majority of which have segregation ratio close to 1:1. The SNP markers across the whole gene are being genotyped in the whole population to map the rusty-specific virulence gene in DW5.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Ameen, G., Kariyawasam, G., Shi, G., Friesen, T.L., Faris, J.D., Ali, S., Rasmussen, J.B., Liu, Z.H. (2017) Molecular manipulation of the mating-type system and development of a new approach for characterizing pathogen virulence in Pyrenophora tritici-repentis. Fungal Genetics and Biology, 109:16-25.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Kariyawasam, G.K, Liu, Z.H. (2017) Developing a bi-parental fungal population and mapping of the genetic locus conditioning Ptr ToxC production in Pyrenophora tritici-repentis. In: Proceeding of 29th Fungal Genetics Conference at Asilomar, Pacific Grove,CA, March 14-19, 2017, pp293 abstract #579F. Poster presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Liu, Z.H. (2017) Development of a new approach to further characterize pathogen virulence mechanisms in the wheat-tan spot disease system. In: Dothideomycetes Comparative Genomics workshop, Oral presentation.
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Progress 02/15/16 to 02/14/17
Outputs
Target Audience:The research results from this grant have been disseminated by the PD and graduate students at national and regional scientific meetings, and seminars, including the NIFA-AFRI project directory meeting in Washington DC, June 30-July 1, 2016, the 28th Fungal Genetics Conference in Pacific Grove, March 17-22, 2015, invited talk at South Dakota State University, December 7, 2015 and departmental seminar at North Dakota State University, Oct 21, 2016. These meetings and seminars included fungal geneticists and biologists, plant geneticists and biologists, graduate students and other professionals. Changes/Problems:Because of sex incomplibilty between virulence race and avirulent race, we cannot develop population between them. We will select a progeny from the developed population which do not produce all three effectors and cause no or low level of disease and will use it to cross with race 2 isolate to develop population for mapping genetic locus encoding additional new effectors. What opportunities for training and professional development has the project provided?Two Ph.D. students, Gayan Kanishka Kariyawasam and Jingwei Guo, in the Department of Plant Pathology, North Dakota State University are being trained in fungal genetic and biology from this grant. Two undergraduate students, Gabrielle Valenzuela and Kristen Almen majoring in agronomy and environmental science, respectively, at NDSU has been trained in molecular genectics and pathology. How have the results been disseminated to communities of interest?The research results from this grant have been disseminated by the PD and graduate students at national and regional scientific meetings, and invited and departmental seminars. What do you plan to do during the next reporting period to accomplish the goals?We will continue to genotype and phenotype the two fungal populations, including 86-124×DW5 and AR CrossB10×86-124 and will use the data to identify genomic region associated with the Ptr ToxC production and new virulence factors.
Impacts
What was accomplished under these goals?
For objective 1), we have created heterothallic fungal strain with one of mating type gene deleted in isolates 86-124 (race 2), DW5 (race 5), AR CrossB10 (new race), L13-192L2 (race 4), and by using the heterothallic strains, the fungal populations have been successfully developed from the crosses of 86-124×DW5, AR CrossB10×DW5, AR CrossB10×86-124 with the number of progeny at 118, 164, and 144, respectively. Because AR CrossB10 produces Ptr ToxC, the populations AR CrossB10×DW5 and AR CrossB10×86-124 will be used in genetic mapping of fungal gene(s) responsible for the Ptr ToxC production. However, the crosses between AR CrossB10 × L13-192L2 did not yield any functional pseudothecia suggesting incompatibility between races 1 and 4. For objective 2), we have started genotyping and phenotyping of the three developed populations and they are at various stages. A considerable work of genotyping and phenotyping has been done for 86-124 × DW5 and AR CrossB10 × 86-124 populations. In 86-124 × DW5 population, the segregation ratios of mating type gene, ToxA, and ToxB were 58:60 (MAT1-1:MAT1-2), 45:73 (present vs absent) and 96:22 (present vs absent). The deviation of ToxB segregation ratio from 1:1 may be due to the presence of multiple copies of ToxB gene in DW5, but the significant deviation of the ToxA gene segregation ratio from 1:1 is not expected, which is intriguing. We obtained fungal progenies that have recombinant genotypes with both the ToxA and ToxB gene or neither of them. We have also genotyped the population with 35 SSR markers, the majority of which have segregation ratio close to 1:1. Genotype-by-sequencing has also been conducted on this population using ion-torrent platform, but no valuable marker was obtained probably due to low genome coverage and low polymorphism between the parental isolates which both come from the North American. Twenty-three progenies have been phenotyped onto tan spot differential lines. DW5 produces Ptr ToxB causing chlorosis on 6B662, but it can cause necrosis a number of hexaploid and tetraploid lines. Therefore, we included these lines in phenotyping to map the necrosis-inducing gene in DW5. Virulence pattern of fungal progenies on differential lines Glenlea and 6B662 correlated with the presence of the ToxA and ToxB gene, respectively. However, the size of chlorotic lesions on 6B662 caused by ToxB-containing progenies varied indicating they may contain different copies of the ToxB gene in the genome. In AR CrossB10×86-124, which is being used to identify Ptr ToxC production gene, the phenotyping for Ptr ToxC production has been done and the two pools of Ptr ToxC producing and non-Ptr ToxC producing progenies. The two pools and parental lines are being used to identify SSR markers linked to the genetic locus conferring Ptr ToxC production.
Publications
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