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.

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.

Small RNA and Degradome Deep Sequencing Reveals the Roles of microRNAs in Peanut (Arachis hypogaea L.) Cold Response.

Cold stress is a major environmental factor that affects plant growth and development, as well as fruit postharvest life and quality. MicroRNAs (miRNAs) are a class of non-coding small RNAs that play crucial roles in various abiotic stresses. Peanuts (Arachis hypogaea L.), one of the most important grain legumes and source of edible oils and proteins, are cultivated in the semi-arid tropical and subtropical regions of the world. To date, there has been no report on the role of miRNAs in the response to cold stress in cultivated peanuts. In this study, we profiled cold-responsive miRNAs in peanuts using deep sequencing in cold-sensitive (WQL20) alongside a tolerant variety (WQL30). A total of 407 known miRNAs and 143 novel peanut-specific miRNAs were identified. The expression of selected known and novel miRNAs was validated by northern blotting and six known cold-responsive miRNAs were revealed. Degradome sequencing identified six cold-responsive miRNAs that regulate 12 target genes. The correlative expression patterns of several miRNAs and their target genes were further validated using qRT-PCR. Our data showed that miR160-ARF, miR482-WDRL, miR2118-DR, miR396-GRF, miR162-DCL, miR1511-SRF, and miR1511-SPIRAL1 modules may mediate cold stress responses. Transient expression analysis in Nicotiana benthamiana found that miR160, miR482, and miR2118 may play positive roles, and miR396, miR162, and miR1511 play negative roles in the regulation of peanut cold tolerance. Our results provide a foundation for understanding miRNA-dependent cold stress response in peanuts. The characterized correlations between miRNAs and their response to cold stress could serve as markers in breeding programs or tools for improving cold tolerance of peanuts.