Source: CORNELL UNIVERSITY submitted to
HIGH RESOLUTION MAPPING OF GENES FOR RESISTANCE TO MULTIPLE DISEASES IN CORN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0216115
Grant No.
(N/A)
Project No.
NYC-153418
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2008
Project End Date
Sep 30, 2011
Grant Year
(N/A)
Project Director
Nelson, RE, J.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Plant Pathology
Non Technical Summary
Northern leaf blight (NLB), and Anthracnose stalk rot (ASR) are among the most important corn diseases in New York, and these diseases also constrain corn production at the national and international levels. Gray leaf spot (GLS) is a problem in several regions of the world, including North America and Africa. The most cost-effective and sustainable way to manage diseases is to incorporate broad-spectrum resistance into corn inbreds and hybrids. Our group focuses on the genetics of disease resistance in corn: we are identifying chromosomal regions and genes that control resistance. This work is aimed at facilitating the efficient incorporation of superior alleles for disease resistance into corn hybrids, breeding lines and populations through MAS. To achieve this, it is critical to precisely map resistance loci and identify the best alleles at those loci. We will take advantage of a breakthrough genetic resource in the corn research community, recently developed by Ed Buckler (Cornell University) and colleagues, to identify genes that confer quantitative disease resistance. Rather than implicating a large genetic region carrying hundreds of genes, we expect to identify the exact genes that provide useful levels of resistance, which will greatly facilitate the utilization of those genes in breeding. The broad diversity of the germplasm base of the mapping population resource will allow for the identification of previously unknown genes and gene variants providing resistance. Through one year of trials to date, we have identified several chromosomal regions that provide resistance to NLB, and one chromosomal region associated with resistance to both NLB and ASR. Through the identification of causal nucleotide polymorphisms for resistance within genes, we can create perfect markers; markers that are not just tightly linked to the resistance allele, but actually fundamental to it. The complementary nature of the NAM, NIL and association mapping populations allows us to combine approaches to further resolve genomic regions down to the causal nucleotide polymorphism. Simultaneously, the high resolution of QRLs will increase our understanding of the genes underlying resistance mechanisms in corn, providing insights into the biology of host-pathogen interactions. This research will build a bridge within the Cornell Research Community, between the Maize Genetics group lead by Ed Bucker, who developed the large genetic resource, and Corn Breeding Program, run by Margaret Smith. The specific outcomes of this project are to identify QRL for resistance to NLB, GLS, and ASR with sufficiently high resolution to allow efficient MAS for these QRL in applied breeding. The identification and characterization of these genes allow corn breeding programs at Cornell University and elsewhere (including in the private sector) to efficiently select for the genes, making more stably resistant inbreds and hybrid cultivars.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011510108070%
2011510116030%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1160 - Pathology; 1080 - Genetics;
Goals / Objectives
Our overall goal is to decipher quantitative resistance in maize for diseases important statewide and nationally, with implications for understanding the more basic nature of quantitative disease resistance, as well breeding for quantitative disease resistance in corn. By utilizing several types of populations, such as the NAM population, and several more traditional fine-mapping populations for specifically targeted regions, we aim to resolve the causal nucleotide polymorphism down to the effective gene and marker. Our more specific objectives are: 1. to map loci associated with quantitative resistance to northern leaf blight (NLB) and gray leaf spot (GLS) to a high level of resolution in the Nested Association Mapping (NAM) population; 2. To genetically dissect the above-mentioned quantitative resistance loci (QRL) of particular interest using the linked genetic resources of the NAM, association mapping panel and near-isogenic lines; specifically targeting the identification of the causal genes underlying at least two QRL regions (one each on chromosomes 1 and 8.06); 3. To characterize selected QRL for their spectrum of resistance against multiple pathogens, and more specifically to determine whether outstanding QRL for anthracnose stalk rot (ASR) and NLB in bin 6.05 are associated with one or more genes; 4. To initiate marker-assisted selection (MAS) for outstanding alleles for NLB, GLS, and ASR in the corn breeding program. The output of this work has both a basic and applied nature. By resolving QRL from the NAM in NLB and GLS down to small genomic regions, and genes, we learn more about host-pathogen interaction as well as advance our knowledge regarding quantitative resistance genes. We also plan to isolate and resolve the causal factors for the QRL and the qualitative Htn1 gene located in chromosome 8.06. We will have identified the QRL genes on 1.02 and 1.06, which appear to have differing entryways into the plant during infection (Chia-Lin Chung, personal communication). The QRL on 6.05 will be resolved to delineate between the NLB and ASR resistance loci, and whether they are unique identities, or indeed an individual event conferring multiple disease resistance. Through the identification of the causal polymorphisms, we can create the most efficient marker assisted selection system, which should aid in the goal of pyramiding QRL genes for cultivar development.
Project Methods
Phenotyping the NAM population: We will evaluate the NAM population described above for resistance NLB and GLS. 1) Screening trials for resistance to NLB will be conducted for three years at Aurora NY with the 2nd and 3rd years (2008 and 2009 environments) conducted with 170 lines/population use a randomized incomplete block design. Field trials for NLB will be artificially inoculated with a New York corn isolate during the 6th week with a spore suspension of conidia at 2,000 spores/plant. Pathogen-cultured sorghum kernels will also be placed in the whole to provide additional inoculum. Disease severity will be rated on a scale of 1-100 (% diseased leaf area) basis. Efficient recording of data will be facilitated by a bar-code system. Upon completion of the three years of evaluation, best linear unbiased estimates of line performance for NLB will be determined using ASREML. SAS program GLM select will be used to select a best fit linear model using a selection threshold of p-value = 4x10-3. QRL mapping will also be conducted using inclusive composite interval mapping. 2) The NAM population will be evaluated for resistance to GLS for three years (2008- 2010) at Blacksburg, VA, in collaboration with Dr. Erik Stromberg of USDA-ARS. A subset of the NAM population (150 RILs/population) will be planted in a randomized incomplete block design and will be naturally inoculated due to the site's history of established heavy GLS pressure. Data collection and analysis will be conducted as mentioned above, except that lines will be rated for disease severity using a disease index of 1-5 in 0.1 increments at four points throughout the disease season. Phenotyping additional targeted populations: 1) A subset of the ~2,600 individual F2 plants that are segregating around the Htn1 locus at 8.06 will be grown in replicated F2:3 rows during 2009 and 2010 in Aurora, NY for additional phenotypic and marker-phenotype validation. 2) DK888-derived F10 lines will be generated in the field in 2008 to be grown out and evaluated in 2009 and 2010, as necessary, to increase resolution for the QRL in the 8.06 region. 3) TBBC3 F3:4 individuals carrying recombination events for two NLB QRLs in bins 1.02 and 1.06 QRLs will be subjected to inoculation and phenotypic assessment in 2009. 4) A large CML52-derived F10 population segregating for the region(s) of interest within 6.05 will also be generated in 2008, for further phenotypic screening for NLB and ASR in 2009 and as necessary. For all the populations listed above, the gene(s) associated with the trait will be determined by break-point analysis. Once the region has been delimited to a small genomic segment, we will utilize the maize genome to identify candidate genes, next generation sequencing to bulk sequence these regions within our materials, and use the nucleotide variation and LD in the maize diversity panel to further resolve the region. We will develop useful, efficient, perfect or causal markers using the state of the art technologies for the use in the corn breeding program led by our collaborator M. Smith for marker assisted selection.

Progress 10/01/08 to 09/30/11

Outputs
OUTPUTS: Data management: A database was developed in Microsoft Access to track our field nursery, raw phenotyping and genotyping data, seed stocks, as well as pathogen stocks. Association mapping: Using the 282-line maize diversity panel, we used 50,000 SNPs to identify markers associated with resistance to northern leaf blight (NLB) and gray leaf spot (GLS). A comparison as made between the SNP markers associated with disease resistance and the current and previously known NLB QTL for confirmation of results. Nested Association Mapping (NAM): Datasets were collected on the resistance of the NAM population for two diseases. A third year of NLB data was collected for 4,630 NAM lines in 2009, and the results were published. The second and third year NAM datasets were collected in 2009 and 2010 for GLS, and a manuscript is currently in preparation. Using joint linkage mapping, QTL were identified for both diseases. Candidate genes were identified by genome-wide association mapping using 1,100 anchored SNP markers across the genome. An additional 1.6 million HapMapv.1 imputed SNP markers were used to identify the putative genes at QTL in the NAM population for NLB and GLS.QTL designated qNLB1.02(Tx303) and qNLB1.06(Tx303) were characterized by fine mapping, histopathological analysis for NLB, and inoculation with a range of pathogens. Through the BC5F2 generation, fixed recombinants were identified using molecular markers and phenotyped for resistance to NLB, Stewart's wilt, and flowering time in Aurora NY. Expression of the candidate gene (Rik) and race specificity of the QTL was tested. Seed for Uniform Mu knock out mutants of Rik in a W22 background were selfed. For NLB1.06(Tx303), two candidate polymorphisms within the interval were identified from association mapping and nested association mapping. The qNLB1.03(Ts2) locus was identified by association mapping, which implicated the Tasselseed2 (Ts2) gene in resistance to NLB. QTL analysis was conducted in F4 (2009) and F5 (2010) families to confirm that NLB resistance segregated at the locus. Mutant analysis was conducted to further confirm association mapping findings. A ts2 mutant was found to have enhanced susceptibility to NLB, was also phenotyped for resistance to SLB and GLS. A segregating family of Ds mutants in the tasselseed1 (ts1) gene was phenotyped for resistance to NLB in Aurora, NY. For B68Htn1(8.06), fine mapping of two loci was conducted from the F4 to F6 generation. Two loci present at the locus were separated and analyzed using SNP markers and field inoculation. For GLS, heterogeneous inbred families (HIF) and segregating families were developed and tested to confirm and fine-map four selected QTL. The following loci were fine-mapped for GLS: qGLS1.05, qGLS2.09, qGLS1.05, and GLS6.05. PARTICIPANTS: Rebecca Nelson, P.I., Scientific Director, McKnight Foundation Collaborative Crop Research Program, Professor in Departments of Plant Pathology and Plant-Microbe Biology, and Plant Breeding and Genetics. Judith Kolkman, Research Support Specialist, qNLB1.03 (ts2), ts1, and finemapping of Htn @ bin 8.06 in maize genome. Jacqueline Benson, graduate student, GLS resistance in maize NAM population. Ashlina Chin, undergraduate employee, summer 2011. L. Michelle Fisher, undergraduate employee, spring 2011. Ariel Fialko, undergraduate employee, June 1 -July 31, 2011. Chris Mancuso, undergraduate employee, summer 2011. Tiffany Jamann, graduate student, fine-mapping of q1.02 and q1.06 in maize. Jesse Poland, graduate student until Dec 2010, GWAS and NLB resistance in NAM population, fine-mapping of q1.06 in maize. J. Benson attended and presented a poster for the 2009 Maize Genetics Conference. In 2010, she developed a presentation for the Northeastern Corn Improvement Conference in Lancaster, PA. She also genotyped Kenyan breeding lines for maize in Nairobi, Kenya in 2010. In 2011, she attended the Meeting of the American Society of Plant Biologists where she presented a poster and received the Pioneer Fellowship Award. J. Kolkman made poster presentations at the 2009, 2010 and 2011 Maize Genetics Conferences. TARGET AUDIENCES: The Nelson lab website (http://www.plantpath.cornell.edu/Labs/Nelson_R/index.html) is continually updated with various interest to a broad range of audiences, including protocols, manuscripts, and information on plant disease. Our lab trains a large group of diverse individuals at the graduate and undergraduate levels. We are responsible for training students who are from NY State, elsewhere in the US, and from other countries. Our research team includes women, African Americans, Asian and African students. Additionally, we have reached out to the school age community of rural NY through participation in a science-based mentorship. Sara Foreman, a senior at Rondout Valley High School was provided the opportunity to work aside Jacqueline Benson in the effort to further elucidate mechanisms underlying host resistance to gray leaf spot of maize primarily using image analysis. Her final project report was submitted for participation in the Intel National Science Fair. As part of our outreach/dissemination efforts, graduate student J. Poland gave a consulting workshop (McKnight Foundation Collaborative Crop Research Program: East and Horn of Africa Community of Practice) in Ethiopia in 2009 as a way to expand utilization of high throughput technologies via bar-coding and database management. Graduate student Jacqueline Benson spent month in Kenya in YEAR to contribute to application of her findings in the context of the Kenyan maize improvement program. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Using NAM, 29 QTLs were identified for NLB and 16 QTL were identified for GLS. While no epistatic interactions were identified for NLB, four two-way allelic interactions were detected for GLS. We fine-mapped 8 QTL: four each for NLB and GLS. Leading candidate genes identified through association mapping implicated jasmonic acid pathways for NLB and anthocyanin/carotenoid pathways for GLS. SNPs identified using association mapping in the maize diversity panel included SNPs in 1.02, 1.03, 1.06, and 6.05. qNLB1.02(Tx303) was found to limit invasion of the plant vasculature, protecting maize from both NLB and Stewart's wilt. The resistant allele reduced disease by over 30%. Its effect was race non-specific with respect to races 0, 1, 23, and 23N of NLB. qNLB1.02 and qStw1.02 were narrowed to 3.41 Mb. The fine-mapping region coincides a SNP in the RIK gene identified by association mapping. RIK is up-regulated in response to NLB, but was not differentially expressed between the near isogenic lines. A major finding is that Tasselseed genes, Ts1 and Ts2, appear to have a dual role in sex determination and disease resistance. Two mutants in the Ts2 gene at qNLB1.03, ts2-ref and ts2-N2409, were found to be susceptible to NLB. The ts2-ref mutant had no effect on resistance to SLB or GLS. Linkage disequilibrium (LD) broke down rapidly within the gene. The identified SNP, ts2.4, and a non-synonymous mutation 10 bp downstream, are in tight LD and the non-synonymous SNP had a stronger association with susceptibility. A ts1:Ds mutant was also found to be susceptible to NLB. Tasselseed1 (Ts1) co-localizes with a NAM QTL.qNLB1.06(Tx303): The NLB and Stewart's wilt fine mapping regions were narrowed to a 5.92 Mb region containing 88 genes. A candidate region of 400 Kb, containing 8 genes, was identified based on genome-wide nested association mapping and genome-wide association mapping using the diversity panel.qNLB6.05: A significant SNP was identified for NLB resistance through association mapping and located within the QTL interval. B68Htn1(8.06): Analysis of the segregating classes within the 450 F4 lines indicated that there are two genes in the Htn differential complex: umc2210 with a major effect locus (~50% AUDPC), and umc1149, a recessive modifier (~5% AUDPC) of the umc2210 locus. The analysis of a heterozygous inbred family (HIF) for GLS confirmed three of loci as contributing significantly to resistance in bins 1.04, 2.09 and 4.05, which span regions of 15 Mb, 5 Mb and 137 Mb, respectively. The resistant allele in bin 1.04 conferred a reduction in disease of 12%. Those in bins 2.09 and 4.05 conferred a reduction of 23% and 8.4%, respectively. qGLS6.05(Mo17) was fine-mapped to 29Mb. qGLS1.05(CML228) was fine-mapped to 11Mb containing 337 genes with an average recombination frequency of 0.236 centimorgans (cM)/Mb. qGLS2.09(CML333) was fine-mapped to 4Mb containing 290 protein coding genes. qGLS4.05(Ki11), a centromeric QTL, was highly strongly associated with resistance (LOD of 270). The low recombination frequency of 0.05cM/Mb was an obstacle to fine mapping; this region is predicted to be 140Mb in size and to contain 7,500 genes.

Publications

  • Belcher AR, J.C. Zwonitzer, JS Cruz, MD Krakowsky, CL Chung, R. Nelson, C. Arellano, and P.J. Balint-Kurti. 2011. Analysis of quantitative disease resistance to southern leaf blight and of multiple disease resistance in maize, using near-isogenic lines. Theor Appl Genet. (online).
  • Chung CL, J. Poland, K. Kump, J. Benson, J. Longfellow, E. Walsh, P. Balint-Kurti, and R. Nelson. 2011. Targeted discovery of quantitative trait loci for resistance to northern leaf blight and other diseases of maize. Theor Appl Genet. 123(2):307-26
  • Wisser RJ, J.M. Kolkman, M.E. Patzoldt, J.B. Holland, J. Yu, M. Krakowsky, R.J. Nelson, P.J. Balint-Kurti. 2011. Multivariate analysis of maize disease resistances suggests a pleiotropic genetic basis and implicates a GST gene. PNAS 108(18):7339-44.
  • Poland, J., P. Bradbury, E. Buckler, and R. Nelson. 2011. Genome-wide Nested Association Mapping of Quantitative Resistance to Northern Leaf Blight in Maize. PNAS 108(17):6893-6898.
  • Poland, J.A., and R.J. Nelson. 2011. In the eye of the beholder: the effect of rater variability and different rating scales on QTL mapping. Phytopathology 101(2):290-298.


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: NAM datasets: A dataset was collected on the resistance of the Nested Association Mapping (NAM) population (n=3,900 RILs) to Gray Leaf Spot (GLS) in a field nursery in Blacksburg VA in 2009. Datasets on this material in VA were also collected for plant and ear height, DTA, row count and quality. Heterogeneous inbred families (HIF) were developed and tested to confirm and further fine-map selected Quantitative Trait Loci (QTL) for GLS Genome wide nested association mapping: Mapping results were obtained for NAM using datasets from three years of Northern Leaf Blight (NLB) and one year of GLS data. Candidate genes were identified by genome-wide association mapping using 1,106 anchored SNP markers across the genome. An additional 1.6 million HapMapv.1 imputed SNP markers were used to resolve genic contributions to QTL in the NAM population. Association mapping: Using the maize diversity panel, 4,000 SNPs were used to identify SNPs associated with NLB in the 282 maize diversity panel. A dataset was produce that identified the genomic regions of the top 10 SNPs conferring resistance to NLB. The SNP markers were compared to current and previously known NLB QTL for confirmation of results. Outputs for specific loci: (1.) qNLB1.02(Tx303): Additional near isogenic lines (NILs) were developed from the Tx303/B73 BC5F2 population. These were selfed in a winter nursery in Argentina. During the summer 2010, 244 fixed recombinants were evaluated for resistance to NLB, Stewart's wilt and rust in Aurora NY. (2) qNLB1.03(Ts2): The Tasselseed2 (Ts2) gene identified using Mixed Linear Models association mapping was evaluated to confirm association mapping findings. A ts2 mutant in a W22 inbred genetic background was obtained and evaluated for resistance to NLB in replicated experiments in Aurora NY, Ithaca NY, and NC. A family segregating for the ts2 mutant based on the 106E was evaluated for resistance to NLB. (3.) qNLB1.06(Tx303): We developed 240 recombinant Tx303/B73 lines via self pollination in the winter nursery. During the summer of 2010, 195 fixed recombinant lines were evaluated for resistance to NLB and Stewart's Wilt in the Aurora field nursery. (4.) B68Htn1(8.06): Additional SNP markers were identified between the two parental inbreds, B68 and B6HtN, using the 6,000 Illumina Maize (SNP) chip. F3 lines from the B68/B68HtN population were genotyped with the umc2210 and umc1149 SSR markers that segregate for resistance to NLB in the B68/B68Htn population. In order to differentiate the effect of each of the two loci individually in bin 8.06, 7 lines homozygous for either the resistant umc2210 or umc1149 alleles and with corresponding susceptible alleles at the second loci were identified and evaluated for resistance to NLB in a replicated experiment in Aurora, NY. An additional 450 F4s were identified and evaluated for NLB in a replicated in complete block design. All lines were self-pollinated to produce F5 families for additional studies. Approximately 1300 lines that were heterozygous at both umc2210 and umc1149 advanced to the F5 generation through self-pollination for future fine-mapping studies in this project. PARTICIPANTS: Rebecca Nelson, P.I., Scientific Director, McKnight Foundation Collaborative Crop Research Program, Associate Professor in Departments of Plant Pathology and Plant-Microbe Biology, and Plant Breeding and Genetics Judith Kolkman, Research Support Specialist, qNLB1.03 (ts2) and finemapping of Htn @ bin 8.06 in maize genome Jacqueline Benson, graduate student, GLS resistance in maize NAM population Chia-Lin Chung, postdoctoral fellow until June 2010, finemapping of q8.06 and q6.05 in maize Sara Heins, undergraduate student employee, summer 2009 Ashlina Chin, undergraduate employee, summer 2010 Tiffany Jamann, graduate student, fine-mapping of q1.02 and q1.06 in maize Jesse Poland, graduate student until Dec 2010, GWAS and NLB resistance in NAM population, finemapping of q1.06 in maize Professional development opportunities were presented to T. Jamann, J. Poland, and J. Kolkman in the form of poster presentation at a professional meeting. J. Benson made a presentation for the Northeastern Corn Improvement Conference (delivered by E. Stromberg) and she was able to extend her knowledge base with a trip to Nairobi to work with the KARI Maize Breeder, James Gethi. TARGET AUDIENCES: A presentation on Jaci Benson's work on the "Genetics of Gray Leaf Spot Resistance," was presented by Erik Stromberg (in her place) at the 64th Northeastern Corn Improvement Conference held February 18-19, 2010. This information was presented to a group of industry and corn breeders who would use this information to further the efforts in breeding resistance varieties. Also, Jaci Benson endeavored on a trip to BecA in Nairobi to genotype breeding materials developed by James Gethi, the KARI Maize Breeder, that were known to segregate from gray leaf spot, northern leaf blight, and maize streak virus. The Nelson lab website (http://www.plantpath.cornell.edu/Labs/Nelson_R/index.html) is continually updated with various interest to a broad range of audiences, including protocols, manuscripts, and information on plant disease. Our lab also trains a large group of diverse individuals at the graduate and undergraduate levels. We are responsible for training students who are from NY state, the US, including women, African Americans, Asian and African students. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
NAM Evaluation & Analysis: Using genome wide nested association mapping, 30 and 21 SNPs across the maize genome were associated with resistance to NLB and GLS, relating to 30 and 21 unique QTLs for resistance to NLB and GLS respectively. Analysis of a HIF family for GLS confirmed three loci as contributing significantly to disease resistance. The confirmed loci are in bins 1.04, 2.09 and 4.05, spanning regions of 15 Mb, 5 Mb and 137 Mb, respectively. SNP markers associated with these loci received the greatest LOD scores from the model selection QTL analysis. The resistant allele in bin 1.04 conferred a reduction in disease of 12%. Those in bins 2.09 and 4.05 conferred a reduction of 23% and 8.4%, respectively. Association mapping using the maize diversity panel: Although the initial series of 4,000 SNP markers should only be considered preliminary, we could identify several significant SNP associations with this set. The top 10 SNPs identified using mixed linear model association mapping identified a number of candidate genes including: a GST gene (bin 7.02), RIK (bin 1.02), Tasselseed2 (bin1.03), and an uncharacterized mRNA sequence (bin 6.05). qNLB1.02(Tx303): The qNLB1.02 and qStw1.02 region has been narrowed to 3.41 Mb, spanning from 24.32 Mb to 27.73 Mb. This region contains about 78 genes. qRust1.02 currently spans 4.71 Mb and does not c-localize with the qNLB1.02 and qStw1.02 region. A significant diversity panel SNP was identified at 26.12 Mb in the RIK gene from the genome wide association mapping. The fine-mapping region coincides with the location of this SNP. This gene encodes for a protein named RIK, that has K homology motif that binds RNA, as well as a proline-rich feature. qNLB1.03: A synonymous SNP in the Ts2 (alcohol dehydrogenase) gene was identified from association mapping in the diversity panel. Sequence analysis in the maize diversity panel identified a linked and significantly associated non-synonymous SNP that is a valid functionally causal SNP. Analysis of both ts2 mutants indicated that this gene is involved in defense response. In both the ts2-ref mutant in the W22 inbred background, and the segregating ts2 (ts2-106E) family, the knocked-out ts2 plants had significantly more NLB. qNLB1.06(Tx303): The NLB and Stewart's wilt resistance co-localize to a 5.92 Mb region. This region contains about 88 genes. Three candidate SNPs in the larger 15 Mb interval have been identified using genome wide nested association mapping. One protein kinase is in the refined fine mapping interval of 6 Mb. qNLB6.05: A significant SNP was identified for resistance to NLB through association mapping analysis using the maize diversity panel that was found to be located within the NIL interval previously identified from previous fine-mapping studies. There is also a QTL for GLS in the bin 6.05 region. B68Htn1(8.06): Distribution of SSR markers in bin 8.06 indicate segregation distortion in the region between umc2210 and umc1149. Analysis of B68 and B68HtN with the Maize (SNP) chip provided for six SNPs near the umc2210 and six SNP markers near the umc1149 SSR that will be utilized in the 8.06 region.

Publications

  • Benson, J., The Maize Diversity Project, E. Stromberg, and R.J.. Nelson. 2010. Genetics of Gray Leaf Spot Resistance. 64th Northeastern Corn Improvement Conference, February 18-19, 2010, Lancaster, Pennsylvania.
  • Chung, C.-L. 2010. Mapping and Characterization of Loci Conditioning Quantitative Disease Resistance in Maize. PhD Dissertation, Cornell University, pp. 224.
  • Chung, C., T. Jamann, J. Longfellow, and R. Nelson. 2010. Characterization and fine-mapping of a resistance locus for northern leaf blight in maize bin 8.06. Theoretical and Applied Genetics. 121(2)205-227.
  • Chung, C., J. Longfellow, E. Walsh, Z. Kerdieh, G. Van Esbroeck, P. Balint-Kurti, and R. Nelson. 2010. Resistance loci affecting distinct stages of fungal pathogenesis: use of introgression lines for QTL mapping and characterization in the maize - Setosphaeria turcica pathosystem. BMC Plant Biology 10:103.
  • Jamann, T.M., C. Chung, J.A. Poland, R.J. Nelson. 2010. Progress towards the Positional Cloning of Three Northern Leaf Blight Quantitative Disease Resistance Loci Maize Genetics Conference Abstracts. 52:P226.
  • Kolkman, J., R. Wisser, C.-L. Chung, J.C. Glaubitz, P. Balint-Kurti, R.J. Nelson. 2010. Association and linkage analysis implicates the Ts2 region in resistance to NLB in maize. Maize Genetics Conf. Abstracts 52:P176.
  • Poland, J. 2010. The Genetic Architecture of Quantitative Disease Resistance in Maize. PhD Dissertation, Cornell University, pp.216.
  • Poland, J., The Maize Diversity Project, R.J. Nelson. 2010. The genetic architecture of complex disease resistance in plants: Northern Leaf Blight in maize. Maize Plant & Animal Genomes XVIII Conf., P346.


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The Nested Association Mapping (NAM) population (n=4,631maize Recombinant Inbred Lines(RILs)) was scored for Northern Leaf Blight (NLB) at the Muskgrave Research Farm in Aurora NY in 2009. NAM was inoculated with NLB and screened at three intervals for Diseased Leaf Area (DLA) and Days to Anthesis (DTA). NAM (n=3,900 RILs) was planted in Blacksburg VA in 2009 and infected with Gray Leaf Spot (GLS) via heavy natural inoculation, scored for GLS, as well as plant and ear height, DTA, row count and quality. Leaves were collected from NAM and scanned with a color-coded legend as a reference control for lesion pigment color. Analysis from two years of NLB and one year of GLS data from NAM was conducted using General Linear Model in SAS (GLM Select). Lines were identified that segregated within families for three GLS QTLs and were planted, genotyped and selfed in the summer of 2009 in Aurora. qNLB1.02(Tx303): A Tx303/B73 BC5F2 population was grown and selfed in Florida. 3,456 plants of the BC5F3 population were planted in Aurora in 2009, genotyped to determine recombinants at bin 1.02, and selfed for generation advancement. Forty-one Near Isogenic Lines (NILs) homozygous in this region were evaluated in a replicated trial and inoculated with NLB as mentioned previously. Plants were scored and DLA. qNLB1.06(Tx303): 4,080 Tx303/B73 BC4F4 lines were planted in Aurora in 2009 and genotyped with markers at bin 1.06. The Tx303/B73 RIL population from NAM was also reanalyzed individually for QTL. qNLB6.05(CML52): Three CML52/B73 F7, 20 F8, and 13 F9 selected NILs segregating for bin 1.06 or 6.05 were evaluated for resistance to NLB and Stewart's wilt at Aurora in 2009. qNLB8.06(DK888): Nineteen SNP markers within the ~4Mb introgression region were developed and a total of 85 F11 NILs for qNLB8.06(DK888) were derived from selected lines capturing different breakpoints. F11 NILs were evaluated for NLB IP and lesion type at Aurora in 2009. F1 and F2 populations were developed in the greenhouse by crossing the Ht2 and Htn1 differential lines with the F9 NILs carrying DK888 or S11 alleles. B68Htn1(8.06): F2 lines from a B68/B68Htn cross were genotyped with SSRs to determine the target 8.06 interval. 1,859 F3 lines were scored in Aurora in 2009 for IP, DTA, and DLA. A database system was been created via Microsoft Access that tracks our field nursery, raw phenotyping and genotyping data, seed stocks, as well as pathogen stocks. Conference presentations are listed below: Benson, JM, Poland, JA, Maize Diversity Project, Nelson, RJ, Stromberg, EL. 2009. Nested Association Mapping of Gray Leaf Spot Resistance in Maize. Maize Genetics Conf. Abstracts 51:P214. Poland, JA, Chung, C-L, Wisser, RJ, Balint-Kurti, PJ, Kump, KL, Benson, JM, Kolkman, JM, The Maize Diversity Project, Nelson, RJ. 2009. Genetic Dissection Of Quantitative Disease Resistance In Maize. Plant & Animal Genomes XVII Conf., San Diego, CA: W105: Challenge Program. Poland, JA, The Maize Diversity Project, Nelson, RJ. 2009. Nested Association Mapping Of Northern Leaf Blight Resistance In Maize Plant & Animal Genomes XVII Conf., San Diego, CA: P332: Maize, Sorghum, Millet, Sugar Cane, and related. PARTICIPANTS: R.J. Nelson, P.I., Scientific Director, McKnight Foundation Collaborative Crop Research Program, Associate Professor in Departments of Plant Pathology and Plant-Microbe Biology, and Plant Breeding and Genetics Jacqueline Benson, graduate student, GLS resistance in maize NAM population Kelly Bulkeley, administrative assistant for the McKnight Foundation Collaborative Crop Research Program Chia-Lin Chung, graduate student, finemapping of q8.06 and q6.05 in maize Ariel Falko, undergraduate student employee, summer 2009 Sara Heins, undergraduate student employee, summer 2009 Tiffany Jamann, graduate student, finemapping of q1.02 in maize Judith Kolkman, Research Support Specialist, finemapping of Htn @ bin 8.06 in maize genome Oliver Ott, undergraduate student employee, winter nursery, Dec/Jan 08/09 Jesse Poland, graduate student, NLB resistance in NAM population, finemapping of q1.06 in maize TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Analysis of the 2007/08 NAM data for resistance to NLB using GLM Select (SAS) identified 21 Quantitative Trait Loci (QTL) across the genome. Many of these localized to targeted regions near previously identified QTL. Three of these QTL were at novel regions of the genome. Analysis of the 2008 NAM data for resistance to GLS using GLM Select (SAS) of the NAM population identified 10 QTLs across the maize genome. Heterozygous RILs were identified at several GLS QTLs, including QTLs at 4.05, 2.09, and 1.04 for future fine-mapping. qNLB1.02(Tx303): 1,239 Tx303/B73 BC5F3 recombinant plants were identified in the 1.02 region from the B73/Tx303 NIL mapping population. Based on the genotyping of the mapping population, the region containing our QTL of interest is delimited to at 13.4 Mb (17 cM) region. qNLB1.06(Tx303): From the 3,166 Tx303/B73 BC4F4 plants genotyped with 3 SNP markers, 874 plants were found to have recombinant genotypes in the three marker region. Approximately 234 lines had recombinants in the left-flanking introgression interval that overlapped with the NAM Tx303/B73 RIL population QTL results. The 234 lines were selfed for generation advance. qNLB6.05(CML52): Previous HIF analysis identified two QTL affecting multiple diseases at bins 1.06 and 6.05 in the same families derived from the F5 line from the CML52/B73 cross. The CML52-derived QTL at bin 1.06 confers resistance to NLB and Stewart's wilt, and the CML52 allele at bin 6.05 confers resistance to NLB and ASR. qNLB8.06(DK888): Allelism analysis suggested that qNLB8.06DK888 is identical, allelic, or closely linked and functionally related to Ht2. The resistance conditioned by qNLB8.06 was incompletely dominant and varied in effectiveness depending upon allele and/or genetic background. Analysis of the F11 DK888-derived NILs narrowed the locus to a region of 460 kb, spanning 143.92-144.38 Mb on the B73 physical map. Out of 12 annotated genes in the region, three candidate genes including two encoding protein kinases and one encoding a protein phosphatase were identified. B68Htn1(8.06): IP distribution of the B68/B68Htn F2 population did not suggest a single gene segregating for resistance. Genotyping of F2 B68/B68Htn population with SSRs in the 8.06 region in maize showed that more than one QTL confers resistance to NLB. Stepwise multiple regression of the F2 population with SSRs identified 2 loci in the 8.06 region that contributed independently to resistance, a small effect locus near marker umc1149 and a large effect locus near marker umc2210. The umc1149 locus has been implicated in previous NLB studies, and the umc2210 locus is near the DK888 putative Ht2 locus. As part of our outreach efforts, graduate student J. Poland gave a consulting workshop (McKnight Foundation Collaborative Crop Research Program: East and Horn of Africa Community of Practice) in Africa as a way to expand utilization of high throughput technologies via barcoding and database management. As well, R.J. Nelson gave a talk at Colorado State University, entitled Quantitative disease resistance in the cereals on October 18th, 2008 in the Department of Bioagricultural Sciences and Pest Management.

Publications

  • Poland JA, Balint-Kurti PJ, Wisser RJ, Pratt RC, Nelson RJ. 2009. Shades of gray: the world of quantitative disease resistance. Trends Plant Sci. 14(1):21-9.
  • Benson, JM, Poland, JA, Maize Diversity Project, Nelson, RJ, Stromberg, EL. 2009. Nested Association Mapping of Gray Leaf Spot Resistance in Maize. Maize Genetics Conf. Abstracts 51:P214.
  • Poland, JA, Chung, C-L, Wisser, RJ, Balint-Kurti, PJ, Kump, KL, Benson, JM, Kolkman, JM, The Maize Diversity Project, Nelson, RJ. 2009. Genetic Dissection Of Quantitative Disease Resistance In Maize. Plant & Animal Genomes XVII Conf., San Diego, CA: W105: Challenge Program (http://www.intl-pag.org/17/abstracts/W12_PAGXVII_105.html)
  • Poland, JA, The Maize Diversity Project, Nelson, RJ. 2009. Nested Association Mapping Of Northern Leaf Blight Resistance In Maize Plant & Animal Genomes XVII Conf., San Diego, CA: P332: Maize, Sorghum, Millet, Sugar Cane, and related (http://www.intl-pag.org/17/abstracts/P05d_PAGXVII_332.html)