Abstract
Aflatoxin, produced by Aspergillus flavus, is hazardous to health of humans and livestock. The lack of information about large effect QTL for resistance to aflatoxin accumulation is a major obstacle to employ marker-assisted selection for maize improvement. The understanding of resistance mechanisms of the host plant and the associated genes is necessary for improving resistance to A. flavus infection. A suppression subtraction hybridization (SSH) cDNA library was made using the developing kernels of Mp715 (resistant inbred) and B73 (susceptible inbred) and 480 randomly selected cDNA clones were sequenced to identify differentially expressed genes (DEGs) in response to A. flavus infection and map these clones onto the corn genome by in-silico mapping. A total of 267 unigenes were identified and majority of genes were related to metabolism, stress response, and disease resistance. Based on the reverse northern hybridization experiment, 26 DEGs were selected for semi-quantitative RT-PCR analysis in seven inbreds with variable resistance to aflatoxin accumulation at two time points after A. flavus inoculation. Most of these genes were highly expressed in resistant inbreds. Quantitative RT-PCR analysis validated upregulation of PR-4, DEAD-box RNA helicase, and leucine rich repeat family protein in resistant inbreds. Fifty-six unigenes, which were placed on linkage map through in-silico mapping, overlapped the QTL regions for resistance to aflatoxin accumulation identified in a mapping population derived from the cross between B73 and Mp715. Since majority of these mapped genes were related to disease resistance, stress response, and metabolism, these should be ideal candidates to investigate host pathogen interaction and to reduce aflatoxin accumulation in maize.
Highlights
Aspergillus flavus is an endemic fungus that is responsible for ear rot disease and aflatoxin contamination in maize (Widstrom, 1996)
The objectives of this study are to identify differentially expressed genes (DEG) using suppression subtraction hybridization (SSH), analyze their expression, validate in a set of resistant and susceptible inbreds, localize the DEGs on the linkage map constructed in an F2:3population of the cross B73 × Mp715 (Dhakal et al, 2016), and identify potential candidate genes involved in host-plant resistance for future studies
Zein-alpha 19C2 precursor (P1B10) and various acetyl CoA derivatives, which were highly expressed in our experiment, are involved in biosynthetic and metabolic pathways (Kelly et al, 2012; Wang et al, 2013). All these studies along with our findings suggest that carbohydrate metabolism related genes play important role in host plant resistance against A. flavus in maize
Summary
Aspergillus flavus is an endemic fungus that is responsible for ear rot disease and aflatoxin contamination in maize (Widstrom, 1996). Consumption of aflatoxin contaminated grains can be harmful for both human and domestic. Candidate Genes for Aflatoxin Resistance in Corn animals. Aflatoxins are the most widely studied mycotoxins due to its ability to suppress human and animal immune systems and induce liver cancer (Hawkins et al, 2015). Aflatoxin contamination drastically reduces grain value leading to significant economic loss worldwide. Many countries around the world have established rules and regulations for minimizing aflatoxin contamination in foods and feed (FAO, 2004). The U.S Food and Drug Administration (USFDA, 2008) has banned the interstate commerce and consumption of the maize grain contaminated with more than 20 ng/g-1 for aflatoxin
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