Genetic dissection of fatty acid components in the Chinese peanut (Arachis hypogaea L.) mini-core collection under multi-environments

Abstract

Peanut (Arachis hypogaea L.) is an important source of edible oil and protein for human nutrition. The quality of peanut seed oil is mainly determined by the composition of fatty acids, especially the contents of oleic acid and linoleic acid. Improving the composition of fatty acids in the seed oil is one of the main objectives for peanut breeding globally. To uncover the genetic basis of fatty acids and broaden the genetic variation in future peanut breeding programs, this study used genome-wide association studies (GWAS) to identify loci associated with target traits and developed diagnostic marker. The contents of eight fatty acid components of the Chinese peanut mini-core collection were measured under four environments. Using the phenotypic information and over one hundred thousand single nucleotide polymorphisms (SNPs), GWAS were conducted to investigate the genetics basis of fatty acids under multi-environments. Overall, 75 SNPs were identified significant trait associations with fatty acid components. Nineteen associations were repeatedly identified in multiple environments, and 13 loci were co-associated with two or three traits. Three stable major associated loci were identified, including two loci for oleic acid and linoleic acid on chromosome A09 [mean phenotypic variation explained (PVE): 38.5%, 10.35%] and one for stearic acid on B06 (mean PVE: 23%). According to functional annotations, 21 putative candidate genes related to fatty acid biosynthesis were found underlying the three associations. The allelic effect of SNP A09-114690064 showed that the base variation was highly correlated with the phenotypic variation of oleic acid and linoleic acid contents, and a cost-effective Kompetitive allele-Specific PCR (KASP) diagnostic marker was developed. Furthermore, the SNP A09-114690064 was found to change the cis-element CAAT (-) in the promoter of ahFAD2A to YACT (+), leading dozens of times higher expression level. The enhancer-like activity of ahFAD2A promoter was identified that was valuable for enriching the regulation mechanism of ahFAD2A. This study improved our understanding on the genetic architecture of fatty acid components in peanut, and the new effective diagnostic marker would be useful for marker-assisted selection of high-oleic peanut breeding.

Fig 1. Overview of the significant associations identified for the fatty acid components (P < 4.73×10⁻⁷).

(A) Distribution and overlap of identified associated loci for fatty acid traits under “E1-E4” environments. (B) Profile of associations for individual traits or co-localized traits. The vertical column diagram shows the number of the associated loci for individual or multiple traits; the interactive plot shows that these traits identified same associated loci; the horizontal column diagram shows the number of associated loci for each trait. E1 refers to Nanchong in 2015; E2 refers to Wuhan in 2015; E3 refers to Nanchong in 2016; E4 refers to Wuhan in 2016.

Fig 2. GWAS association loci for oleic acid, linoleic acid and stearic acid in the Chinese peanut mini-core collection under four environments.

(A) Manhattan plots and quantile-quantile plots for oleic acid (C18:1). Negative log₁₀(P) values from a genome-wide scan are plotted against position on each of twenty chromosomes. The horizontal dashed lines indicate the genome-wide significance threshold (-log₁₀4.73×10⁻⁷ = 6.33). (B) Manhattan plots and quantile-quantile plots for linoleic acid (C18:2). (C) Manhattan plots and quantile-quantile plots for stearic acid (C18:0). The horizontal coordinates of quantile-quantile plots represented expected -log₁₀(P), and the vertical coordinates of quantile-quantile plots represented observed -log₁₀(P).

Fig 3. Genetic analysis of oleic acid and linoleic acid on A09 and validation of the diagnostic marker.

(A) Take the Manhattan plots of A09 chromosome for oleic acid (C18:1) in E1 environment as an example. (B) The diagram of SNP (G-to-A) in the promoter region of ahFAD2A. (C) The allelic effect at A09-114690064 for oleic acid (C18:1) and linoleic acid (C18:2) under four environments. For each trait, the boxes with GG alleles and AA alleles were significant different according to Tukey’s Multiple Comparison Test (P < 0.05) (D) The partial sequence diagrams included the diagnostic locus of the SNP in the promoter of ahFAD2A after amplifying by PCR and sequencing. The diagnostic SNP locus changed the cis-element of CAAT (-) to enhancer-like module YACT (+). (E) qRT–PCR result of ahFAD2A in seeds at stage 7. (F) Phenotypic differences between accessions carrying different alleles of the SNP A09-114690064 of two extreme trait groups of peanut. P<0.001, Student’s t-test. (G) Scatter plots using KASP marker genotyping.