Genome-wide identification and characterization of NF-Y gene family in peanut (Arachis hypogaea L.)

Nuclear factor Y (NF-Y) gene family consists of NF-YA, NF-YB and NF-YC subfamilies. Many members of NF-Y family have been involved in plant development processes, phytohormone signaling and tolerance to stresses in Arabidopsis and other plant species. However, little attention has been given in peanut. A total of 33 AhNF-Y genes (AhNF-Ys) were identified and distributed on 16 chromosomes. A phylogenetic analysis indicated that NF-Y genes prossessed highly conservatism in different plants. Gene duplication analyze indicated that only segmental duplication were detected. The abiotic stress-related regulatory elements analysis showed that AhNF-Ys, except for AhNF-YB6, contained at least one abiotic stress response element. With RNA-seq data, the tissue/organ-specific expression and differential expression profiling under salt stress were analyzed, indicating that six selected AhNF-Y gene may play potential roles in the regulation of salt stress response. qRT-PCR results suggested that these AhNF-Y genes also responded to osmotic, ABA (Abscisic Acid) and SA (Salicylic acid) stresses. AhNF-Y gene phylogenetic relationship, chromosomal gene duplication on the expansion, and abiotic stress/hormone-related regulatory in promoter of AhNF-Y gene The expression profiles in various and of during by RNA-seq method. In addition, using the RNA-seq and quantitative real-time PCR (qRT-PCR) methods, we analyzed the expression profiles of AhNF-Y genes under salt stress, and identified several candidate genes responsive to abiotic stress and hormonal treatment.


Result
Identification and analysis of AhNF-Y genes By using BLASTP and HMMER software, the amino acid sequences of Arabidopsis and rice NF-Ys were used to search the peanut genome database. Pfam, NCBI-CDD and SMART were used to confirm the NF-Y domain. Finally, 14 NF-YA genes, 10 NF-YB genes and 9 NF-YC genes were identified as peanut NF-Ys ( AhNF-Ys). All these genes were named on the base 6 of the exact position on chromosome. The results of sequence alignment indicated that AhNF-YA certain conserved domains genes. Gene characteristics, including gene names, gene IDs, chromosomal locations, open reading frame (ORF) lengths, exon numbers, amino acid (AA) numbers, molecular weights (MW) and the isoelectric points (pI) were showed in Table 1. AhNF-Y proteins contained 171 (AhNF-YB2) to 492 (AhNF-YA12) amino acid. The MW of AhNF-Ys ranged from 18.88 kDa (AhNF-YB2) to 55.45 kDa (AhNF-YA12), and the pI values were bewteen 5.19 (AhNF-YC9) and 9.64 (AhNF-YA5).

Phylogenetic analysis of AhNF-Y genes
To investigate the evolutionary relationships of NF-Y genes in plants, an un-rooted phylogenetic tree of NF-Y complete protein sequences from Arabidopsis, rice, peanut and human was constructed using MEGA 7. The unrooted maximum likelihood phylogenetic tree was generated from 33 AhNF-Ys, 33AtNF-Ys, 28 OsNF-Ys and 3 NF-Y_HSs. The NF-Y proteins were divided into 3 subfamilies. As indicated in Fig. 1, the members of NF-Y protein family exhibit high homology. In NF-YA subfamily, AhNF-YA1 and A8 exhibit highest homology with AtNF-YA1 and therefore they were orthologous proteins. In NF-YB subfamily, AhNF-YB4/9 and AtNF-YB2 belong to orthologous proteins. Besides, AhNF-YC3/9 share highest similarity with AtNF-YC1. This highly conservatism suggests that the NF-Y proteins in different plant species might share similar function.

Gene structure and motif analysis
The cultivated peanut is an allotetraploid containing of A and B subgenome. With multiple sequences alignment (Figs. 2) and phylogeny evolution analysis (Fig. 2a), 7 pairs of homologous AhNF-YA genes, 5 pairs of homologous AhNF-YB genes and 2 pairs of homologous AhNF-YC genes were identified, respectively (Table 2).
Gene Structure Display Server 2.0 was used to analyze and visualize the structures of NF-7 Y genes in peanut. As shown in Fig. 2a and 2b, AhNF-Ys share high similarity in number and length of introns when the amino acid sequence similarity of two genes was the highest. Comparing the members of the 3 subfamilies, the members of AhNF-YA subfamily contain a larger number of introns. Except for AhNF-YC1, the intron numbers of AhNF-YC subfamily were generally at a low level.
The MEME analysis tool was used to predict the conserved motifs in AhNF-Y genes ( Fig.2c and Additional file 6). Motif 7, 8 were only identified in AhNF-YA subfamily. Motif 5 was observed in each member of AhNF-YA subfamily except for AhNF-YA7 and A14. Motif 2, 4 and 11 were unique to AhNF-YB subfamily. Motif 3, 6 and 10 were existed only in AhNF-YC subfamily. These results indicated that NF-Y is a kind of highly conservative gene family in peanut. Moreover, motif 16 was found only in AhNF-YB3 and B8, and motif 9 and 17 were both identified in 4 AhNF-Y genes (AhNF-YA2, A3, A9 and A10). These specific motifs may result in some functional differences among AhNF-Y genes.

Chromosomal distribution and gene duplication of AhNF-Ys
The 33 AhNF-Y genes were unevenly distributed on 16 chromosomes except chr2, 5, 12 and 15 (Table 1 These results suggested that AhNF-Y genes may involve in many different abiotic stresses.

Tissue/organ-specific expression analysis of AhNF-Ys
To determine the expression patterns of AhNF-Y genes, we used a published RNA-seq data, which covered gene expression profiles of 22 tissues throughout the entire life cycle of peanut [ 57 ]. Based on the expression characteristics, the tissue/organ expression profiles of AhNF-Ys were classified into four categories Fig. 5 . Group 1 contained 3 genes (AhNF-YA3, A10 and C6 ), which showed low expression level in most tissues except seed. Group 2 comprised 8 genes, which were hardly detected or only at low levels in all tissues and organs. The third group included only 2 genes (AhNF-YC1 and C8), which expressed extremely high, specifically in seed developmental stages. Group 4 was composed of the other AhNF-Ys. These genes represented higher expression levels compared with genes belong to Group 2 and display tissue/organ-specific expression patterns. For example, AhNF-YA1 and A8 exhibited root-specific expression; AhNF-YB4, B9 and C3 showed preferential expression in leaves; AhNF-YB2 and B7 were expressed highly only in nodule.
The above results indicated that most AhNF-Y genes were constitutively expressed.
Moreover, the tissues and organs-specific expression of some AhNF-Y genes suggests that they may play different roles in different tissues and growth stages peanut.

Expression analysis of AhNF-Y genes under salt treatment
Using the high through put RNA-seq data of AhNF-Y genes, a heatmap was established to analyze the response to salt stress.  reached peak at about 2 h except for AhNF-YB4. In addition, all these genes were significantly responded to SA tress. All above results indicated that these 6 AhNF-Y genes responded to osmotic, ABA and SA stress with distinctive expression patterns. Discussion NF-Y gene family, as a transcription factor family, is present in almost all plant species.
However, the function in cultivated peanut is little known. In our study, 33 AhNF-Y genes were identified from the cultivated peanut genome database.
In our study, four pairs of AhNF-Y genes were detected as segment duplications, but no tandem duplication event was found, suggesting that only the segmental duplication contribute to the expansion of AhNF-Y gene family. And the remaining AhNF-Y genes may evolve in an early divergence time or be obtained from gene translocation.
To further investigate the putative function of AhNF-Ys, the RNA-seq data was analyzed. Previous study of their ortholog PvNF-YB7 in Phaseolus vulgaris was consistent with our speculation [ 48 ]. The homologous genes AhNF-YB2 and AhNF-YB7 which had similar expression profiles were specifically expressed in nodule, thereby suggesting that their functions may be related to the formation of roots and nodules. Moreover, the expression levels of AhNF-YB4 and AhNF-YB9 were relatively higher in leaf and stem than root. These 2 genes may play similar roles with their orthologs AtNF-YB3 and AtNF-YB2, which were concern with the response to the endoplasmic reticulum stress and/or in the promotion of flowering in

Plant Materials and Growth Conditions
Mature seeds of Arachis hypogasa L. cultivar Fenghua 2 (Spanish type) were used in this research. The seeds were germinated on distilled water-wet degreasing cotton in seedling-15 raising disks. These disks were placed at 26℃ in darkness for 3 days, and then the germinated seeds were exposed to long-day conditions (LD; 16h light and 8h dark cycle).

Stress treatment, total RNA extraction and revers transcription
To analysis expression pattern of AhNF-Y genes, two-week old seedlings were treated with nutrient solution containing 200mM NaCl, 20% (w/v) mannitol, 100mM ABA and 100mM SA, respectively. Leaf and root of seedlings treated with NaCl were harvested at 0h and 16h.
These samples were used for transcriptome and qRT-PCR analysis. Leaves of seedlings treated with mannitol were collected at 0h, 2h, 4h, 6h, 8h, 12h and 24h. For ABA and SA treatment, leaves of seedlings were harvested at 0h, 1h, 2h, 4h, 6h and 8h. All samples were frozen immediately in liquid nitrogen for RNA extraction. The experiments were conducted with three independent biological replicates.       qRT-PCR profiles of 6 AhNF-Y genes in response to mannitol. 14 days-old seedling leaves were sampled at 0, 2, 4, 8, 12 and 24 h under a 16-h light/8-h dark cycle.
Bars reflect the means ± SD of three replicates. Asterisks indicate the corresponding gene significantly up-or down-regulated compared with untreated control (**P < 0.01 and *P < 0.05, Student's t-test).
37 Figure 8 qRT-PCR profiles of 6 AhNF-Y genes in response to ABA and SA. 14 days-old