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Identification of Two Novel Peanut Genotypes Resistant to Aflatoxin Production and Their SNP Markers Associated with Resistance.

ABSTRACT: Aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) are the most common aflatoxins produced by Aspergillus flavus in peanuts, with high carcinogenicity and teratogenicity. Identification of DNA markers associated with resistance to aflatoxin production is likely to offer breeders efficient tools to develop resistant cultivars through molecular breeding. In this study, seeds of 99 accessions of a Chinese peanut mini-mini core collection were investigated for their reaction to aflatoxin production by a laboratory kernel inoculation assay. Two resistant accessions (Zh.h0551 and Zh.h2150) were identified, with their aflatoxin content being 8.11%-18.90% of the susceptible control. The 99 peanut accessions were also genotyped by restriction site-associated DNA sequencing (RAD-Seq) for a genome-wide association study (GWAS). A total of 60 SNP (single nucleotide polymorphism) markers associated with aflatoxin production were detected, and they explained 16.87%-31.70% of phenotypic variation (PVE), with SNP02686 and SNP19994 possessing 31.70% and 28.91% PVE, respectively. Aflatoxin contents of accessions with "AG" (existed in Zh.h0551 and Zh.h2150) and "GG" genotypes of either SNP19994 or SNP02686 were significantly lower than that of "AA" genotypes in the mean value of a three-year assay. The resistant accessions and molecular markers identified in this study are likely to be helpful for deployment in aflatoxin resistance breeding in peanuts.

SUBMITTER: Yu B

PROVIDER: S-EPMC7150746 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Identification of Two Novel Peanut Genotypes Resistant to Aflatoxin Production and Their SNP Markers Associated with Resistance.

Yu Bolun B Jiang Huifang H Pandey Manish K MK Huang Li L Huai Dongxin D Zhou Xiaojing X Kang Yanping Y Varshney Rajeev K RK Sudini Hari K HK Ren Xiaoping X Luo Huaiyong H Liu Nian N Chen Weigang W Guo Jianbin J Li Weitao W Ding Yingbin Y Jiang Yifei Y Lei Yong Y Liao Boshou B

Toxins 20200301 3

Aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) are the most common aflatoxins produced by Aspergillus flavus in peanuts, with high carcinogenicity and teratogenicity. Identification of DNA markers associated with resistance to aflatoxin production is likely to offer breeders efficient tools to develop resistant cultivars through molecular breeding. In this study, seeds of 99 accessions of a Chinese peanut mini-mini core collection were investigated ...[more]

PMID: 32121605

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Project description:BackgroundAflatoxin contamination caused by Aspergillus flavus is a major constraint to peanut industry worldwide due to its toxicological effects to human and animals. Developing peanut varieties with resistance to seed infection and/or aflatoxin accumulation is the most effective and economic strategy for reducing aflatoxin risk in food chain. Breeding for resistance to aflatoxin in peanut is a challenging task for breeders because the genetic basis is still poorly understood. To identify the quantitative trait loci (QTLs) for resistance to aflatoxin contamination in peanut, a recombinant inbred line (RIL) population was developed from crossing Zhonghua 10 (susceptible) with ICG 12625 (resistant). The percent seed infection index (PSII), the contents of aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) of RILs were evaluated by a laboratory kernel inoculation assay.ResultsTwo QTLs were identified for PSII including one major QTL with 11.32-13.00% phenotypic variance explained (PVE). A total of 12 QTLs for aflatoxin accumulation were detected by unconditional analysis, and four of them (qAFB1A07 and qAFB1B06.1 for AFB1, qAFB2A07 and qAFB2B06 for AFB2) exhibited major and stable effects across multiple environments with 9.32-21.02% PVE. Furthermore, not only qAFB1A07 and qAFB2A07 were co-localized in the same genetic interval on LG A07, but qAFB1B06.1 was also co-localized with qAFB2B06 on LG B06. Conditional QTL mapping also confirmed that there was a strong interaction between resistance to AFB1 and AFB2 accumulation. Genotyping of RILs revealed that qAFB1A07 and qAFB1B06.1 interacted additively to improve the resistance to both AFB1 and AFB2 accumulation. Additionally, validation of the two markers was performed in diversified germplasm collection and four accessions with resistance to aflatoxin accumulation were identified.ConclusionsSingle major QTL for resistance to PSII and two important co-localized intervals associated with major QTLs for resistance to AFB1 and AFB2. Combination of these intervals could improve the resistance to aflatoxin accumulation in peanut. SSR markers linked to these intervals were identified and validated. The identified QTLs and associated markers exhibit potential to be applied in improvement of resistance to aflatoxin contamination.

Project description:IntroductionPeanut 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.ObjectivesThis 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.MethodsResistance 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.ResultsThe 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.ConclusionOur findings would provide insights of aflatoxin production resistance mechanisms and laid meaningful foundation for further breeding programs.

Project description:Peanut is susceptible to Aspergillus flavus infection, and the consequent aflatoxin contamination has been recognized as an important risk factor affecting food safety and industry development. Planting peanut varieties with resistance to aflatoxin contamination is regarded as an ideal approach to decrease the risk in food safety, but most of the available resistant varieties have not been extensively used in production because of their low yield potential mostly due to possessing small pods and seeds. Hence, it is highly necessary to integrate resistance to aflatoxin and large seed weight. In this study, an RIL population derived from a cross between Zhonghua 16 with high yield and J 11 with resistance to infection of A. flavus and aflatoxin production, was used to identify quantitative trait locus (QTL) for aflatoxin production (AP) resistance and hundred-seed weight (HSW). From combined analysis using a high-density genetic linkage map constructed, 11 QTLs for AP resistance with 4.61-11.42% phenotypic variation explanation (PVE) and six QTLs for HSW with 3.20-28.48% PVE were identified, including three major QTLs for AP resistance (qAFTA05.1, qAFTB05.2 and qAFTB06.3) and three for HSW (qHSWA05, qHSWA08 and qHSWB06). In addition, qAFTA05.1, qAFTB06.3, qHSWA05, qHSWA08 and qHSWB06 were detected in multiple environments. The aflatoxin contents under artificial inoculation were decreased by 34.77-47.67% in those segregated lines harboring qAFTA05.1, qAFTB05.2 and qAFTB06.3, while the HSWs were increased by 47.56-49.46 g in other lines harboring qHSWA05, qHSWA08 and qHSWB06. Conditional QTL mapping indicated that HSW and percent seed infection index (PSII) had no significant influence on aflatoxin content. Interestingly, the QT 1059 simultaneously harboring alleles of aflatoxin content including qAFTA05.1 and qAFTB05.2, alleles of PSII including qPSIIB03.1, qPSIIB03.2, and qPSIIB10 and alleles of HSW including qHSWA05, qHSWB06, qHSWA08 had better resistance to A. flavus infection and to toxin production and higher yield potential compared with the two parents of the RIL. The above identified major loci for AP resistance and HWS would be helpful for marker-assisted selection in peanut breeding.

Project description:Aflatoxin contamination caused by Aspergillus flavus in peanut (Arachis hypogaea) including in pre- and post-harvest stages seriously affects industry development and human health. Even though resistance to aflatoxin production in post-harvest peanut has been identified, its molecular mechanism has been poorly understood. To understand the mechanism of peanut response to aflatoxin production by A. flavus, RNA-seq was used for global transcriptome profiling of post-harvest seed of resistant (Zhonghua 6) and susceptible (Zhonghua 12) peanut genotypes under the fungus infection and aflatoxin production stress.A total of 128.72 Gb of high-quality bases were generated and assembled into 128, 725 unigenes (average length 765 bp). About 62, 352 unigenes (48.43%) were annotated in the NCBI non-redundant protein sequences, NCBI non-redundant nucleotide sequences, Swiss-Prot, KEGG Ortholog, Protein family, Gene Ontology, or eukaryotic Ortholog Groups database and more than 93% of the unigenes were expressed in the samples. Among obtained 30, 143 differentially expressed unigenes (DEGs), 842 potential defense-related genes, including nucleotide binding site-leucine-rich repeat proteins, polygalacturonase inhibitor proteins, leucine-rich repeat receptor-like kinases, mitogen-activated protein kinase, transcription factors, ADP-ribosylation factors, pathogenesis-related proteins and crucial factors of other defense-related pathways, might contribute to peanut response to aflatoxin production. Notably, DEGs involved in phenylpropanoid-derived compounds biosynthetic pathway were induced to higher levels in the resistant genotype than in the susceptible one. Flavonoid, stilbenoid and phenylpropanoid biosynthesis pathways were enriched only in the resistant genotype.This study provided the first comprehensive analysis of transcriptome of post-harvest peanut seeds in response to aflatoxin production, and would contribute to better understanding of molecular interaction between peanut and A. flavus. The data generated in this study would be a valuable resource for genetic and genomic studies on crops resistance to aflatoxin contamination.

Project description:BACKGROUND: Pre-harvest infection of peanuts by Aspergillus flavus and subsequent aflatoxin contamination is one of the food safety factors that most severely impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. Some peanut cultivars with natural pre-harvest resistance to aflatoxin contamination have been identified through field screening. However, little is known about the resistance mechanism, which has slowed the incorporation of resistance into cultivars with commercially acceptable genetic background. Therefore, it is necessary to identify resistance-associated proteins, and then to recognize candidate resistance genes potentially underlying the resistance mechanism. RESULTS: The objective of this study was to identify resistance-associated proteins in response to A. flavus infection under drought stress using two-dimensional electrophoresis with mass spectrometry. To identify proteins involved in the resistance to pre-harvest aflatoxin contamination, we compared the differential expression profiles of seed proteins between a resistant cultivar (YJ-1) and a susceptible cultivar (Yueyou 7) under well-watered condition, drought stress, and A. flavus infection with drought stress. A total of 29 spots showed differential expression between resistant and susceptible cultivars in response to A. flavus attack under drought stress. Among these spots, 12 protein spots that consistently exhibited an altered expression were screened by Image Master 5.0 software and successfully identified by MALDI-TOF MS. Five protein spots, including Oso7g0179400, PII protein, CDK1, Oxalate oxidase, SAP domain-containing protein, were uniquely expressed in the resistant cultivar. Six protein spots including low molecular weight heat shock protein precursor, RIO kinase, L-ascorbate peroxidase, iso-Ara h3, 50 S ribosomal protein L22 and putative 30 S ribosomal S9 were significantly up-regulated in the resistant cultivar challenged by A. flavus under drought stress. A significant decrease or down regulation of trypsin inhibitor caused by A. flavus in the resistant cultivar was also observed. In addition, variations in protein expression patterns for resistant and susceptible cultivars were further validated by real time RT-PCR analysis. CONCLUSION: In summary, this study provides new insights into understanding of the molecular mechanism of resistance to pre-harvest aflatoxin contamination in peanut, and will help to develop peanut varieties with resistance to pre-harvested aflatoxin contamination.

Project description:BackgroundAflatoxin contamination caused by Aspergillus fungi has been a serious factor affecting food safety of peanut (Arachis hypogaea L.) because aflatoxins are highly harmful for human and animal health. As three mechanisms of resistance to aflatoxin in peanut including shell infection resistance, seed infection resistance and aflatoxin production resistance exist among naturally evolved germplasm stocks, it is highly crucial to pyramid these three resistances for promoting peanut industry development and protecting consumers' health. However, less research effort has been made yet to investigate the differentiation and genetic relationship among the three resistances in diversified peanut germplasm collections.ResultsIn this study, the Chinese peanut mini-mini core collection selected from a large basic collection was systematically evaluated for the three resistances against A. flavus for the first time. The research revealed a wide variation among the diversified peanut accessions for all the three resistances. Totally, 14 resistant accessions were identified, including three with shell infection resistance, seven with seed infection resistance and five with aflatoxin production resistance. A special accession, Zh.h1312, was identified with both seed infection and aflatoxin production resistance. Among the five botanic types of A. hypogaea, the var. vulgaris (Spanish type) belonging to subspecies fastigiata is the only one which possessed all the three resistances. There was no close correlation between shell infection resistance and other two resistances, while there was a significant positive correlation between seed infection and toxin production resistance. All the three resistances had a significant negative correlation with pod or seed size. A total of 16 SNPs/InDels associated with the three resistances were identified through genome-wide association study (GWAS). Through comparative analysis, Zh.h1312 with seed infection resistance and aflatoxin production resistance was also revealed to possess all the resistance alleles of associated loci for seed infection index and aflatoxin content.ConclusionsThis study provided the first comprehensive understanding of differentiation of aflatoxin resistance in diversified peanut germplasm collection, and would further contribute to the genetic enhancement for resistance to aflatoxin contamination.

Dataset Information

Identification of Two Novel Peanut Genotypes Resistant to Aflatoxin Production and Their SNP Markers Associated with Resistance.

Publications

Identification of Two Novel Peanut Genotypes Resistant to Aflatoxin Production and Their SNP Markers Associated with Resistance.

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