Identification and application of a candidate gene AhAftr1 for aflatoxin production resistance in peanut seed (Arachis hypogaea L.)

Abstract

Introduction: Peanut is susceptible to infection of Aspergillus fungi and conducive to aflatoxin contamination, hence developing aflatoxin-resistant variety is highly meaningful. Identifying functional genes or loci conferring aflatoxin resistance and molecular diagnostic marker are crucial for peanut breeding.

Objectives: This work aims to (1) identify candidate gene for aflatoxin production resistance, (2) reveal the related resistance mechanism, and (3) develop diagnostic marker for resistance breeding program.

Methods: Resistance to aflatoxin production in a recombined inbred line (RIL) population derived from a high-yielding variety Xuhua13 crossed with an aflatoxin-resistant genotype Zhonghua 6 was evaluated under artificial inoculation for three consecutive years. Both genetic linkage analysis and QTL-seq were conducted for QTL mapping. The candidate gene was further fine-mapped using a secondary segregation mapping population and validated by transgenic experiments. RNA-Seq analysis among resistant and susceptible RILs was used to reveal the resistance pathway for the candidate genes.

Results: The major effect QTL qAFTRA07.1 for aflatoxin production resistance was mapped to a 1.98 Mbp interval. A gene, AhAftr1 (Arachis hypogaea Aflatoxin resistance 1), was detected structure variation (SV) in leucine rich repeat (LRR) domain of its production, and involved in disease resistance response through the effector-triggered immunity (ETI) pathway. Transgenic plants with overexpression of AhAftr1_(ZH6) exhibited 57.3% aflatoxin reduction compared to that of AhAftr1_(XH13). A molecular diagnostic marker AFTR.Del.A07 was developed based on the SV. Thirty-six lines, with aflatoxin content decrease by over 77.67% compared to the susceptible control Zhonghua12 (ZH12), were identified from a panel of peanut germplasm accessions and breeding lines through using AFTR.Del.A07.

Conclusion: Our findings would provide insights of aflatoxin production resistance mechanisms and laid meaningful foundation for further breeding programs.

Keywords: Aflatoxin production resistance; Diagnositic marker; ETI; NB-LRRs; Peanut.

Graphical abstract

Fig. 1

Phenotype distribution in peanut seeds of parental lines and RIL population (A) The dynamic changes of seed aflatoxin content in XH13 and ZH6 after A. flavus inoculation. Values are means ± standard deviations (SD). (B) The picture of XH13 and ZH6 in the 7th day after inoculation. (C) Phenotypic observation and distribution of AFTs in parents and RIL population. The arrows represent the position of two parents for AFTs in RIL population. (D) Phenotypic variability among the RILs selected for development of extreme bulks for aflatoxin content. Based on the three environments phenotyping of RIL population, 10 low aflatoxin content RILs and 10 high aflatoxin content RILs were used to construct resistance and susceptible bulks (RB and SB).

Fig. 2

QTL-mapping for aflatoxin production resistance in peanut seeds (A) ΔSNP-index plot between ZH13 and ZH6 in A07 chromosome. (B) QTLs identified by genetic linkage analysis. The LOD value map of AFTs in whole genome among three environments.

Fig. 3

Fine mapping of AhAftr1 (A) Fine mapping of genomic region for aflatoxin resistance. Up side is the 11 SNP markers used to screen homozygous recombinants. Left side is the graphical genotypes of 11 homozygous recombinants types (G2-G12) and two parent types (G1 for ZH6 and G13 for XH13). “n” represent the number of each homozygous family. Black and white bars represent the chromosome segments from ZH6 and XH13, respectively. Right side is the aflatoxin content (Mean ± SD) for each homozygous family, green colour represent resistance type (the aflatoxin content was significantly lower than G13), red colour represent susceptible type (the aflatoxin content was significantly higher than G1), significant difference are indicated by ** (p < 0.01). (B) The distribution of 13 genes in 103.34 kb candidate genomic region. (C) Diagram of nucleotide polymorphism for AhAftr1. The polymorphic site and relative position are indicated on the coding sequence of AhAftr1. “-” represent deletion. (D) Position of conserved domain of AHAFTR1 and the variation sites of the protein. “-” represent deletion. (E) Three dimensional protein structure of AHAFTR1 predicated by Alpha Flod 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Transgenic experiments and diagnostic marker application (A) Transgenic maize after inoculation. (B) Relative expression of AhAftr1 in transgenic maize lines. (C) Phenotype analysis of transgenic maize lines. a, b, c, d and e represent the significance of differences between different transgenic lines. (D) PCR products of AFTR.Del.A07 amplified in XH13, ZH6 and ZH12. (E) Phenotypic effect of AFTR.Del.A07 in RILs population. (F) Phenotypic effect of AFTR.Del.A07 in 144 Chinese peanut germplasm collection. (G) Phenotypic effect of AFTR.Del.A07 in 62 breeding lines. “AA” represents accessions shown the same genotype with SP. “aa” represent accessions shown the same genotype with RP. “n” represent the number of accessions for each genotype.

Fig. 5

DEG analysis and KEGG pathway for AhAftr1 (A) The number of upregulated (upper/orange bars) and downregulated (lower/green bars) genes in RBT as compared with SBT at each time point after inoculation. (B) KEGG analysis of DEGs. (C) Validation of 4 genes in RNA-seq data by RT-qPCR. Y-axis showed the log2(R/S) between R and S. Positive value indicated up-regulated in R, negative value indicated down-regulated in R. (D) A part of the KEGG pathway for AhAftr1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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