|Wang, X. -|
|Wadl, P. -|
|Wood-Jones, A. -|
|Trigiano, R. -|
|Scruggs, M. -|
|Pilgrim, C. -|
|Baird, R. -|
Submitted to: Mycopathologia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 3, 2012
Publication Date: August 20, 2012
Citation: Wang, X., Wadl, P., Wood-Jones, A., Windham, G.L., Trigiano, R., Scruggs, M., Pilgrim, C., Baird, R. 2012. Characterization of expressed sequence tag-derived simple sequence repeat markers for Aspergillus flavus: emphasis on variability of isolates from the southern United States. Mycopathologia. 174:371–382. Interpretive Summary: The fungus Aspergillus flavus produces aflatoxin in corn grain and is a major problem for growers in the southeastern United States. Isolates of this fungus vary in aflatoxin production and pathogenicity; however, little information is known on the genetic diversity of A. flavus isolates. Molecular techniques utilizing simple sequence repeats (SSRs) developed from an A. flavus expressed sequence tag (EST) database were used to examine the genetic relationships of 96 Aspergillus isolates from numerous host species and geographical regions. The isolates could be divided into six groups. These groups were not related to any visible morphological feature such as colony color or type of overwintering structure such as sclerotial type. There was no apparent grouping of isolates based on aflatoxin production, host species, or geographic origin. A large number of isolates collected from the Mississippi Delta region did not show any unusual grouping except for three isolates which included K32, K55, and 199. We were able to detect a higher level of genetic variability in A. flavus isolates using EST-SSRs compared to previous studies that used genomic SSRs. In addition, using EST-SSRs was a reliable method for separating A. flavus isolates from other Aspergillus species (A. caelatus, A. niger, and A. nomius).
Technical Abstract: Simple Sequence Repeat (SSR) markers were developed from Aspergillus flavus expressed sequence tag (EST) database to conduct an analysis of genetic relationships of Aspergillus isolates from numerous host species and geographical regions, but primarily from the United States. Twenty-nine primers were designed from 362 tri-nucleotide EST-SSR sequences. Eighteen polymorphic loci were used to genotype 96 Aspergillus species isolates. The number of alleles detected per locus ranged from 2 -to -24 with a mean of 8.2 alleles. Haploid diversity ranged from 0.28 to 0.91. Genetic distance matrix was used to perform principle coordinates analysis (PCA) and to generate dendrograms using unweighted pair group method with arithmetic mean (UPGMA). Two principal coordinates explained more than 75% of the total variation among the isolates. One clade was identified for A. flavus isolates (n=87) with the other Aspergillus species (n = 7) using PCA, but five distinct clusters were present when the others taxa were excluded from the analysis. Six groups were noted when the EST-SSR data was compared using UPGMA. However, the latter PCA or UPGMA comparison resulted in no direct associations to host species, geographical region or aflatoxin production. Furthermore there was no direct correlation to visible morphological features such as sclerotial types. The isolates from Mississippi Delta region, which contained the largest percentage of isolates, did not show any unusual clustering except for isolates K32, K55, and 199. Further studies of these three isolates are warranted to evaluate their pathogenicity, aflatoxin production potential, additional gene sequences (eg. RPB2) and morphological comparisons.