Screening Of Peanut Genotypes For Low Temperature Tolerance And Identification Of Low Temperature Responsive Genes

Peanut or groundnut (Arachis hypogaea L.) is one of the most important cash crops in the world. It is also a rich source of edible protein and oil. Temperature is an important factor affecting peanut growth and development and limiting the geographical distribution of the peanut crop. Screening for peanut genotypes with low temperature tolerance and isolation and characterization of low temperature responsive transcripts are of great significance to the development of low temperature tolerant peanut cultivars with high and stable yields and enlarged peanut cultivation in cooler areas.Tolerance to low temperature during germination for fifty-five accessions of peanut germplasm were evaluated when the seeds were imbibed at 2℃for 96h followed by 25℃for 72h. Percentage of the number of seeds with radicals breaking through testa (PSWRBT) and ratio of the length of hypocotyls and radicals to the length of seeds (RHRS) were calculated. Main quality traits of the entries, including oleic acid, linoleic acid, palmitic acid, oil, protein and sucrose content were determined by near-infrared spectroscopy. Relationship between low temperature tolerance of peanut seed during inbibition and individual quality attributes was analyzed. The results showed that under low temperature stress conditions, 3,3,5 and 43 accessions had the PSWRBT of≥80%, 70%~80%, 60%~70% and <60%, respectively; 3,5,3 and 43 accessions had the RHRS of >0.5 , 0.4~0.5, 0.4~0.3 and <0.3, respectively. A4, a valencia type accession with 95% PSWRBT and 1.50 RHRS were identified as highly tolerant. PSWRBT was significantly positively related to RHRS and positively related to oil content; RHRS was significantly positively related to linoleic acid content and negatively related to oleic acid content; neither of them was significantly positively related to 100-seeds weight.To isolate differentially expressed peanut genes responsive to low temperature, by using a PCR-SelectTM cDNA Subtraction Kit, 3 suppression subtractive hybridization (SSH) cDNA libraries, named library A, library B and library C, were constructed for a low temperature tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2℃and 15℃, respectively, for 1h, 6h and 24h. A total of 500 randomly picked cDNA clones from each library were sent for sequencing. Among them 336 from library A, 429 from library B and 466 from library C were successfully sequenced, resulting in 8 unique transcripts (7 contigs and 1 singlets) in library A, 18 unique transcripts (14 contigs and 4 singlets) in library B, and 193 unique transcripts (73 contigs and 120 singlets) in library C. Sequence alignment and gene annotation with BLAST2GO revealed that of these unique transcripts, 7 from library A, 14 from library B and 132 from library C were significantly similar to the sequences in GenBank non- redundant (nr) protein database, which belonged to diverse functional categories including transcriptional regulation, energy metabolism, stress defense, cell structure, cell protection, signal transduction and biosynthesis.Based on the results from sequence alignment and gene annotation, 14 transcripts of interest were further analyzed with RT-qPCR for gene differential expression at normal and low temperature. Indeed, these genes were found expressed differentially in stressed and control peanut seeds (with relative expression ranging from 2.85 to 12.85, as calculated by average of 3 replications using the 2-ΔΔCt algorithms).From the above-mentioned transcripts confirmed by RT-qPCR, 4 were selected in subsequent studies to obtain complete coding sequence using the RACE method. These were LEA, Oleosin, NAC and MYB. The latter two were transcription factors. To the best of our knowledge, thus far there has been no report on MYB in peanut. Sequence analysis showed that the cloned LEA was 1526 bp in length, coding for 144 amino acids, Oleosin was 760 bp in length, coding for 176 amino acids, NAC was 1406 bp in length, coding for 301 amino acids, MYB was 1365 bp in length, coding for 344 amino acids. The physical, chemical properties, domains, activity sites, and secondary and tertiary structures of the proteins encoded by these genes were predicted by bioinformatics tools. The present study paved the way to both functional analysis of low temperature-responsive genes using experimental methods, i.e., gene knock-in and knock-out, and molecular breeding for low temperature tolerance in peanut.