New tools to screen wild peanut species for aflatoxin accumulation and genetic fingerprinting

Abstract

Background: Aflatoxin contamination in peanut seeds is still a serious problem for the industry and human health. No stable aflatoxin resistant cultivars have yet been produced, and given the narrow genetic background of cultivated peanuts, wild species became an important source of genetic diversity. Wild peanut seeds, however, are not abundant, thus, an effective method of screening for aflatoxin accumulation using minimal seeds is highly desirable. In addition, keeping record of genetic fingerprinting of each accession would be very useful for breeding programs and for the identification of accessions within germplasm collections.

Results: In this study, we report a method of screening for aflatoxin accumulation that is applicable to the small-size seeds of wild peanuts, increases the reliability by testing seed viability, and records the genetic fingerprinting of the samples. Aflatoxin levels observed among 20 wild peanut species varied from zero to 19000 ng.g^-1 and 155 ng.g^-1 of aflatoxin B₁ and B₂, respectively. We report the screening of 373 molecular markers, including 288 novel SSRs, tested on 20 wild peanut species. Multivariate analysis by Neighbor-Joining, Principal Component Analysis and 3D-Principal Coordinate Analysis using 134 (36 %) transferable markers, in general grouped the samples according to their reported genomes. The best 88 markers, those with high fluorescence, good scorability and transferability, are reported with BLAST results. High quality markers (total 98) that discriminated genomes are reported. A high quality marker with UPIC score 16 (16 out of 20 species discriminated) had significant hits on BLAST2GO to a pentatricopeptide-repeat protein, another marker with score 5 had hits on UDP-D-apiose synthase, and a third one with score 12 had BLASTn hits on La-RP 1B protein. Together, these three markers discriminated all 20 species tested.

Conclusions: This study provides a reliable method to screen wild species of peanut for aflatoxin resistance using minimal seeds. In addition we report 288 new SSRs for peanut, and a cost-effective combination of markers sufficient to discriminate all 20 species tested. These tools can be used for the systematic search of aflatoxin resistant germplasm keeping record of the genetic fingerprinting of the accessions tested for breeding purpose.

Keywords: Arachis; Aspergillus flavus; Fingerprinting; aflatoxin; groundnut; molecular markers; peanut.

Fig. 1

Left: Percentage of markers from this study, found in the same corresponding chromosomes of A. ipaënsis and A. duranensis (total 193 markers), and their distribution by chromosome. Right: Size of each of the 10 chromosomes of A. ipaënsis and A. duranensis, in proportion to their entire genomes. Arrows indicate the larger size of chromosome three and smaller size of chromosome eight in A. duranensis. Chr: chromosome

Fig. 2

Neighbor-joining dendrogram based on Nei’s standard genetic distance using 134 molecular markers on 20 wild species of peanut. The samples are colored to indicate the overall grouping of species by genome type (dark blue: A, pink: E, grey: K, green: H, light blue: B D F). The same colors were used for each sample in PCA and 3D-PCoA analyses. Only bootstrap values higher than 50 are shown at the nodes. For graphic clarity the species names were shortened, please see List of Abbreviations. Species names are followed by PI, genome type and Section (Ar: Arachis; Er: Erectoides; He: Heteranthae; Pr: Procumbentes; Rh: Rhizomatosae). 98 good-quality markers that discriminated genomes follow the same color code in Additional file 4: Table S4

Fig. 3

Graph of the first two axes from a principal component analysis (PCA) (Top) and 3-Dimention-Principal Coordinate Analysis (3D-PCoA) (Bottom) of 20 wild species of peanut using 134 molecular markers (including SSRs and InDels). The first component explains 16 % and the second 11 % of the total genetic variation; and the first 3 PCoA dimensions explained 34 %, 25 % and 21 % (total 80%) of the genetic variation. For graphic clarity the species names were shortened, please see List of Abbreviations. Color code as in Fig. 2

Fig. 4

Electropherogram of amplicons generated on 20 wild peanut species using marker NPRL_cont01020a, this primer had UPIC score 16, that means 16 allele patterns were observed. X axis is in base pairs, and Y axis is fluorescence level. For graphic clarity the species names were shortened, please see List of Abbreviations

Fig. 5

Percentage of heterozygous loci observed on 20 wild peanut species using 134 molecular markers that were transferable to all the species. For graphic clarity the species names were shortened, please see List of Abbreviations

Fig. 6

Concentration of aflatoxin B₁ and B₂ detected on individual seeds of 16 wild peanut species. Only 16, out of 20 species tested shown viability in in vitro culture, therefore were used in the analysis. Same letters indicate samples not significantly different from each other; samples without letters were not included in the statistical analysis. For graphic clarity the species names were shortened, please see List of Abbreviations