1. Identification of Gene-Based Markers for Resistance to Aflatoxin Accumulation in Corn by Examination of the Plant / Pathogen / Environment Interactions Ashli Brown (PI), Xueyan Shan (Co-PI), Mississippi State University; Dawn S. Luthe (Co-PI), Pennsylvania State University; Collaborators: Paul Williams, Marilyn Warburton, Erik Mylroie, Gary Windham, USDA/ARS Mississippi State University; Daniel Peterson, Mississippi State University; Robert Kemerait, University of Georgia; Baozhu Guo, USDA/ARS Tifton, Georgia; Zhi-Yuan Chen, Louisiana State University; Alisa Huffaker, Eric Schmelz, USDA/ARS Gainesville, Florida Year 2 Year 1

2.  Examination of the plant / pathogen / environment components to better understand the complex host- pathogen interaction  Identify gene-based aflatoxin resistance markers

3. Project Objectives:  1. Identify resistance candidate genes to develop gene-based markers for marker-assisted breeding.  2. Gather a “snapshot” of gene expression and protein expression profiles for corn germplasm lines with varying levels of resistance / susceptibility to aflatoxin accumulation following inoculation with A. flavus strains.

4. Year 2 (2013):  Genomic, proteomic, and metabolomics studies and analysis of results  Screening and validation of candidate genes  Development of DNA markers

5. Gathering gene expression profiles through Real Time qPCR (RT-qPCR) technique

6. AI664980 Va35 Mp313E Gene Expression Levels in kernel samples revealed via RT-qPCR

7. Va35 Mp313E Va35

8. Mp313E TC231674 is a resistance candidate gene found highly expressed in both of the resistant maize inbred lines Mp313E and Mp718

9. A barplot showing comparisons in expression levels of differentially expressed genes (Mp313E vs Va35)

10. Time course profiles for selected genes that highly expressed in the resistant corn inbred line Mp313E

11. Time course profiles for selected genes that highly expressed in the susceptible corn inbred line Va35

12. MP04:85 Va35 MP04:89MP715  Maize lines were inoculated with A. flavus.  Kernel samples were collected at 1, 2, 4, 7, 14, and 21 days after inoculation.  Samples were flash frozen to retain integrity of nucleic acids and other small molecules.

13. MP04:104 Mp719 Mp718 Va35

14. PedigreeHost Plant ResponseTreatmentAlfatoxin (ng/g) Level* Va35SusceptibleInoculated1821 a Uninoculated≈1 Mp04:85SusceptibleInoculated1594 ab Uninoculated≈1 Mp04:89SusceptibleInoculated1236 ab Uninoculated≈1 Mp04:104ResistantInoculated255 c Uninoculated≈1 Mp718ResistantInoculated199 c Uninoculated≈1 Mp719ResistantInoculated53 d Uninoculated≈1 *Means followed by the same letter do not differ at p=0.05 (Fisher’s Protected LSD). Test of significance were performed on transformed value (log(Y+1)). Alfatoxin concentrations in mature kernels of the six maize inbred lines.

15. Gathering a “snapshot” of differentially expressed gene profiles Sample 1-36, 2DAI; 37-72, 7DAI

16. Gathering a “snapshot” of differentially expressed gene profiles Sample 1-36, 2DAI; 37-72, 7DAI

17. Gathering a “snapshot” of differentially expressed gene profiles Sample 1-36, 2DAI; 37-72, 7DAI

18. An example of a gene expressing consistently in all the tested inbred lines Sample 1-36, 2DAI; 37-72, 7DAI

19. Gathering a “snapshot” of differentially expressed gene profiles Sample 1-36, 2DAI; 37-72, 7DAI

20. Another example of a gene expressing consistently in all the tested inbred lines Sample 1-36, 2DAI; 37-72, 7DAI

21. AI664980TC231674 Integrated QTL Map

22. Mp715 Va35 SNP analysis from gene sequences

23. Diversity in gene sequences of AI664980 among corn inbred lines

24. Abundant SNPs (DNA markers) revealed via DNA sequence analysis

26. Indels are found to distinguish between some resistant maize lines (Mp313E, Mp715) and some susceptible maize lines (NC300, T173, B73). (Data from Leigh Hawkins and Marilyn Warburton)

27. Protein Extraction was performed using TCA/acetone precipitation in combination of a phenol extraction. Protein concentration was determined by Bio-Rad protein assay dye reagent at 595 nm Developing kernels from two resistant (Mp719 and Mp715) and two susceptible (Mp04:87 and Va35) maize inbred lines Proteomic Studies

28. Mp179 rep1Mp179 rep2Mp179 rep3 Mp04:87rep1 Mp04:87rep3 Mp04:87rep2 Mp175rep2Mp175rep1Mp175rep3 Va35 rep1 Va35 rep2 Va35 rep3

29. Metabolomics Candidate phytoalexins associated with aflatoxin accumulation RNA sequencing Comparative modeling Single Kernel Aflatoxin LC/MS Method Metabolomics with SPME and GC/MS and GC-QTOF technologies. What’s next: Collaborations will continue in the future.

30. What’s next: Whole genome sequencing of maize inbred lines Mp313E and Mp715 to discover markers associated with resistance to Aspergillus flavus and aflatoxin reduction in maize A newly funded AMCOE project, Xueyan Shan (MSU) (PI), Prasanta Subudhi (LSU), Ashli Brown (MSU), Darrell Sparks (MSU) (Co-PIs)

31. Selected Abstracts and Presentations: Brown A, Sparks D, Williams WP, Rodriguez J, Ward S, Armbrust K. 2012. Monitoring Aflatoxin in Mississippi. Joint IFSH/FRI Mycotoxin Symposium, Rosemont, IL (April 12, 2012). Atkinson C, Pechanova O, Rodriguez J, Sparks D, Brown A. 2012. Rapid Identification of Filamentous Fungi. AOAC Southern Section Annual Meeting, Atlanta, GA. (April 30-May 1, 2012). Reid CX, Johnston CI, Bell K, Larson E, Brown A, Sparks D. 2012. Monitoring Aflatoxin Contamination of Corn in Mississippi. AOAC Southern Section Annual Meeting, Atlanta, GA. (April 30- May 1, 2012). Pechanova O, Pechan T, Rodriguez J, Williams WP, Brown A. 2012. 2D Reference Map of the Aflatoxingenic Fungus Aspergillus flavus. America Society for Mass Spectrometry, Vancouver, Canada, (May 20-24, 2012). Mylroie JE, Shivaji R, Ozkan S, Alpe MN, Windham GL, Williams WP. 2012. Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus strain using Quantitative Real-Time PCR. Corn Utilization and Technology Conference. Indianapolis, IN (June 4-6, 2012). Tiwari A, Williams WP, Shan X. 2012. Comparative Structural Analysis of Maize Homologous Proteins Tentatively Associated with Host Plant Resistance to Aspergillus flavus Infection and Aflatoxin Accumulation. Corn Utilization and Technology Conference. Indianapolis, IN (June 4-6, 2012).

32. Selected Abstracts and Presentations: Alka Tiwari, W. Paul Williams, J. Erik Mylroie, Gary L. Windham, Ashli Brown, Xueyan Shan. ‘Computational analysis of 2D protein gel images for identification of differentially expressed proteins associated with resistance to aflatoxin accumulation in maize’. MCBIOS 2013 Conference, April 5-6, 2013, Columbia, MO. Matthew Asters, W. Paul Williams, Andy Perkins, J. Erik Mylroie, Gary L. Windham, Xueyan Shan. ‘Quantitative RT-PCR Analysis of Maize Genes Associated with Resistance to Aspergillus flavus’. MCBIOS 2013 Conference, April 5-6, 2013, Columbia, MO. S. Kate Thompson, Alka Tiwari, W. Paul Williams, Xueyan Shan. ‘Identification of DNA polymorphisms in maize genes related to Aspergillus flavus resistance’. Undergraduate Symposium, Mississippi State University, Spring 2013. Taylor Clark, Alka Tiwari, W. Paul Williams, Xueyan Shan. ‘Study of Single Nucleotide Polymorphisms (SNPs) in resistant and susceptible maize inbred lines’. Undergraduate Symposium, Mississippi State University, Summer 2013. Alka Tiwari, Doctoral Dissertation, Biochemistry, Molecular Biology, Entomology and Plant Pathology, Title: Genomic and Proteomic Analysis of Maize Inbred Lines. Fowlkes Auditorium – 2:00 – 3:20 P.M. November 5, 2013. Three Minute Thesis, Graduate School, Mississippi State University

33. Selected publications from the Collaborative Team: Kelley RY, Williams WP, Mylroie JE, Boykin DL, Harper JW, Windham GL, Ankala A, Shan X. (2012) Identification of Maize Genes Associated with Host Plant Resistance or Susceptibility to Aspergillus flavus Infection and Aflatoxin Accumulation. PLoS ONE 7(5): e36892. doi:10.1371/journal.pone.0036892 Pechanova O, Pechan T, Rodriguez J, Williams WP, Brown A. (2012) A Two-dimensional Proteome Reference Map of the Aflatoxigenic Fungus Aspergillus flavus. Proteomics. Johnston I, Singleterry R, Reid C, Sparks D, Baldwin B, Hill-Ward S, Williams WP, Brown A. (2012) Fermentation of Aflatoxin Inoculated Corn. Natural Resources. 3(3) DOI10:4236/nr.2012.33017. Semagn K., C. Magorokosho, B.S. Vivek, D. Makumbi, Y. Beyene, S. Mugo, B.M. Prasanna and M.L. Warburton. 2012. Molecular characterization of diverse CIMMYT maize inbred lines from eastern and southern Africa using single nucleotide polymorphic markers. BMC Genomics 13:113, doi:10.1186/1471-2164-13-113. W. Paul Williams, Michael N. Alpe, Gary L. Windham, Seval Ozkan, J. Erik Mylroie Comparison of Two Inoculation Methods for Evaluating Maize for Resistance to Aspergillus flavus Infection and Aflatoxin Accumulation. International Journal of Agronomy Volume 2013, Article ID 972316, http://dx.doi.org/10.1155/2013/972316