Location: Crop Protection and Management Research2011 Annual Report
1a. Objectives (from AD-416)
(1) Develop peanut genomic tools and strategies to elucidate the molecular mechanisms that define crop defense pathways and regulation of resistance to diseases such as tomato spotted wilt virus (TSWV), leaf spots and aflatoxin contamination in peanut. (2) Evaluate corn germplasm that harbors resistance genes that reduce aflatoxin contamination and understand the responding genes and pathways in corn.
1b. Approach (from AD-416)
(1) Replicated laboratory and field screening and evaluation of peanut accessions for disease resistance will be conducted in order to identify the “resistant” germplasm for further genomic studies. The resistant germplasm will be utilized in molecular marker development for marker-assisted breeding. (2) ESTs (expressed sequence tags) will be generated from cDNA libraries constructed from seed and leaf tissues of two genotypes, Tifrunner and GT-C20. EST-derived SSR markers will be developed and peanut oligo-microarray will be produced for gene expression study. (3) Genetic mapping populations (RILs, recombinant inbred lines) will be produced from crosses of Tifrunner and GT-C20, and SunOleic 97R x NC94022. QTL mapping will be conducted for resistance to tomato spotted-wilt virus (TSWV) and leaf spots, and aflatoxin contamination. (4) Microarray experiments will be used to identify candidate genes in corn-Aspergillus flavus and drought stress interactions that are turned on or off during corn kernel development. The candidate genes identified from microarray will be verified or confirmed through real time PCR or other well established methods. Another goal is to develop a macroarray tool (membranes) using these candidate genes from microarray to assess resistance or drought tolerance in corn germplasm for their stability of expression in native crops under environmental conditions (e.g., drought) known to be conducive to aflatoxin contamination. The genes identified in corn kernels also will be applied in searching possible ‘orthologs’ in peanut genome and peanut germplasm.
3. Progress Report
Peanut is vulnerable to a range of diseases, such as tomato spotted wilt virus (TSWV) and early and late leaf spots. The application of biotechnology for improving peanut has been hampered by an inability to visualize sufficient genetic variation and a high-resolution genetic linkage map. The objective of this study was to develop a comparative integrated map from two recombinant inbred line (RIL) populations. A total of 4576 simple sequence repeat (SSR) markers from three sources: published SSR markers, newly developed SSR markers from expressed sequence tags (EST) and from bacterial artificial chromosome (BAC) end-sequences were used for screening polymorphisms. A total of 324 markers were anchored on this integrated map covering 1,352.1 cM. High consistence with other cultivated peanut maps derived from different populations may support this integrated map as a reliable reference map for peanut whole genome sequencing assembling. Using this map, we have detected two Quantitative Trait Loci (QTLs) for resistance to TSWV in these two populations. The total oil and oleic contents have been analyzed and ranged from 38% to 54% of total oil contents and 34% to 84% oleic contents. To characterize SSR markers and genetic relationships within cultivated peanut, a total of 709 SSR markers were collected from public databases and 556 SSRs passed an initial screen and were used to characterize 16 peanut genotypes. Two hundred thirty-five markers showed polymorphisms in these genotypes. The average heterozygosity estimated from these 556 SSRs was 0.225. The average number of alleles per SSR was 2.5. However, 410 SSR markers had only one allele, confirming that diversity of cultivated peanuts is very limited. The genetic relationships are in agreement with the pedigrees and origins of the tested peanut genotypes, indicating that these SSR markers are useful tools for evaluation of genetic diversity in peanuts. To analyze the expression of stress-related genes in different corn inbred with different resistance to aflatoxin contamination, 94 genes were selected to test the differential expression in corn inbred, A638, B73, Lo964, Lo1016, Mo17, Mp313E, and Tex6. Based on the relative-expression levels, the seven maize inbred lines clustered into two different groups. One group included B73, Lo1016 and Mo17, which had higher levels of aflatoxin contamination and lower levels of overall gene expression. The second group which included Tex6, Mp313E, Lo964 and A638 had lower levels of aflatoxin contamination and higher overall levels of gene expressions. A total of six “cross-talking” genes were identified between the two groups. When further subjected to drought stress, Tex6 has more genes up-regulated and B73 has fewer genes up-regulated. The transcription patterns and interactions measured in this study indicate that the resistant mechanism is an interconnected process involving many gene products and transcriptional regulators, as well as environmental factors such as drought and heat.
1. Gene expression and morphological alternation of corn root architecture in response to drought stress. Water-deficit stress tolerance is a complex trait, and water-deficit results in various physiological and chemical changes and exacerbates preharvest aflatoxin contamination. The objective of this study was to characterize the variations in morphology, physiology and gene expression in two contrasting inbred lines, Lo964 and Lo1016, in order to understand the differences in response to water-deficit stress and to breed corn with drought tolerance. The results revealed that Lo964 was less sensitive to water-deficit stress, and had a strong lateral root system and a higher root/shoot ratio in comparison to Lo1016, sensitive to water stress. In response to water-deficit stress by comparing stressed versus watered conditions, abscisic acid (ABA) syntheses were increased in leaves, roots and kernels of both Lo064 and Lo1016, but by different magnitudes. However, in the kernels, ABA levels were increased in all samples in both lines. Indole-3-cetic acid (1AA) was undetectable in the leaves and roots of either genotype regardless of treatments, but increases of 58% and 8% in IAA concentration were observed in 20 days after planting (DAP) kernels, in response to water-deficit stress, respectively. These data support previous observation that Lo964 is more tolerant to water-deficit stress than Lo1016. Further study is needed to confirm if Lo964 has reduced aflatoxin contamination asociated with the drought tolerance in the field in order to utilize the resistant trait in breeding.
2. Variability in field response of peanut lines from the U.S. and China to tomato spotted wilt virus and leaf spots. Tomato spotted wilt, caused by tomato spotted wilt virus (TSWV) and transmitted by thrips, and early leaf spot and late leaf spot are among the most important diseases of peanut in the southeastern United States. The objective of this study was to compare field susceptibility of diverse peanut lines to TSWV and leaf spot pathogens for decision making for selecting lines for mapping population development. In field trials in 2007 and 2008, 22 genotypes were evaluated for reactions to TSWV and leaf spots. Early leaf spot was predominated in both years. There was a near-continuous range of spotted wilt from 18% to 79% for the total spotted wilt incidence rating with any symptoms caused by TSWV. In general, NC94022, ‘Georganic’, C689-6-2, ‘Georgia-01R’, C724-19-25, TifGP-1, C11-154-61, C12-3-114-58, and ‘Tifguard’ were among the most resistant genotypes to TSWV, whereas GTC-20, GTC-9 and PE-2 were the most susceptible ones. Final percentage of defoliation by leaf spots ranged from 10% to 97% for both years. Genotypes C689-2, Georgia-01R, C12-3-114-58, C11-154-61, Tifguard and Georganic showed resistance to leaf spots, whereas ‘NC-6’, ‘Spancross’, GTC-9, GTC-20 and PE-2 are susceptible to leaf spots. These phenotypic disease reaction data can be used in conjunction with genetic characterization of these genotypes for development of recombinant inbred line populations in efforts to develop markers for resistance to TSWV and leaf spots.
Luo, M., Liu, J., Lee, R.D., Scully, B.T., Guo, B. 2010. Monitoring the expression of maize genes in developing kernels under drought stress using oligo-microarray. Journal of Integrative Plant Biology. 52(12):1059-1074.