Fine mapping of a QTL and identification of candidate genes associated with cold tolerance during germination in peanut (Arachis hypogaea L.) on chromosome B09 using whole genome re-sequencing

Abstract

Low temperatures significantly affect the growth and yield of peanuts. Temperatures lower than 12 °C are generally detrimental for the germination of peanuts. To date, there has been no report on precise information on the quantitative trait loci (QTL) for cold tolerance during the germination in peanuts. In this study, we developed a recombinant inbred line (RIL) population comprising 807 RILs by tolerant and sensitive parents. Phenotypic frequencies of germination rate low-temperature conditions among RIL population showed normally distributed in five environments. Then, we constructed a high density SNP-based genetic linkage map through whole genome re-sequencing (WGRS) technique and identified a major quantitative trait locus (QTL), qRGRB09, on chromosome B09. The cold tolerance-related QTLs were repeatedly detected in all five environments, and the genetic distance was 6.01 cM (46.74 cM - 61.75 cM) after taking a union set. To further confirm that qRGRB09 was located on chromosome B09, we developed Kompetitive Allele Specific PCR (KASP) markers for the corresponding QTL regions. A regional QTL mapping analysis, which was conducted after taking the intersection of QTL intervals of all environments into account, confirmed that qRGRB09 was between the KASP markers, G22096 and G220967 (chrB09:155637831-155854093), and this region was 216.26 kb in size, wherein a total of 15 annotated genes were detected. This study illustrates the relevance of WGRS-based genetic maps for QTL mapping and KASP genotyping that facilitated QTL fine mapping of peanuts. The results of our study also provided useful information on the genetic architecture underlying cold tolerance during germination in peanuts, which in turn may be useful for those engaged in molecular studies as well as crop improvement in the cold-stressed environment.

Keywords: QTL; candidate genes; cold tolerance; germination; peanut; whole genome re-sequencing.

Figure 1

Flow chart for population development, high-density genotyping and multi-environments phenotyping.

Figure 2

Phenotypic variation of cold tolerance-related Traits in RILs and their parents. (A) Germination experiments were performed in a growth chamber at a relative humidity of 70% and a temperature of 12 °C for 3 d, then at 2 °C for 3 d, and finally at 25 °C for 3 d to recovery treatment. The parents showed distinct differences in cold tolerance. The radicle lengths of almost all tested Huayu 44’s seeds were greater than their seed lengths. By contrast, the radicle lengths of most DF12’s seeds were either equal to or less than their seed lengths, while some seeds failed to germinate. R, female parent Huayu 44; S, male parent DF12. (B) Frequency distribution for relative germination rate in RIL population at E1, E2, E3, E4 and E5. Instrument-measured traits in all five environments followed normally distributed.

Figure 3

Genome variations and annotations. (A) Circos plot of SNP and InDel distributions. The outer ring indicates the SNP distribution, whereas the inner ring indicates the InDel distribution. (B) Pie charts of SNP annotations. (C) Pie charts of InDel annotation information.

Figure 4

Bin map and heatmap of the RIL population. (A) A total of 2494 bins were inferred from resequencing-based high-quality SNPs. Red indicates DF12’s background, and blue indicates Huayu 44’s background. Yellow indicates heterozygous genotypes and missed genotypes. (B) Genome-wide recombination hotspots in the 20 peanut chromosomes.

Figure 5

High-density genetic map. A high-resolution and high-quality genetic map had been constructed for QTL mapping. The short black lines on linkage groups mean the locus of bin markers.

Figure 6

The germination stage cold-related QTL qRGRB09 is located on chromosome B09. qRGRB09 on chromosome B09 was stably detected in all five environments.

Figure 7

Fine mapping of cold-related QTLs and candidate genes screen. (A) KASP-based regional linkage analysis to narrow the region QTL (qRGRB09) using IciMapping V4.1 in the RIL population (black curve). P16, KASP marker G22096; P17, KASP marker G22097. (B) Candidate genes identified in 216.26 Kb QTL region mapped on chromosome B09. This region was rich in RLKs, tRNA ligase, MYB, BI1-like protein, DnaJ, serine/threonine-protein kinase, and other uncharacterized proteins.