Global Survey, Expressions and Association Analysis of CBLL Genes in Peanut

Abstract

Cystathionine γ-synthase (CGS), methionine γ-lyase (MGL), cystathionine β-lyase (CBL) and cystathionine γ-lyase (CGL) share the Cys_Met_Meta_PP domain and play important roles in plant stress response and development. In this study, we defined the genes containing the Cys_Met_Meta_PP domain (PF01053.20) as CBL-like genes (CBLL). Twenty-nine CBLL genes were identified in the peanut genome, including 12 from cultivated peanut and 17 from wild species. These genes were distributed unevenly at the ends of different chromosomes. Evolution, gene structure, and motif analysis revealed that CBLL proteins were composed of five different evolutionary branches. Chromosome distribution pattern and synteny analysis strongly indicated that whole-genome duplication (allopolyploidization) contributed to the expansion of CBLL genes. Comparative genomics analysis showed that there were three common collinear CBLL gene pairs among peanut, Arabidopsis, grape, and soybean, but no collinear CBLL gene pairs between peanut and rice. The prediction results of cis-acting elements showed that AhCBLLs, AdCBLLs, and AiCBLLs contained different proportions of plant growth, abiotic stress, plant hormones, and light response elements. Spatial expression profiles revealed that almost all AhCBLLs had significantly higher expression in pods and seeds. All AhCBLLs could respond to heat stress, and some of them could be rapidly induced by cold, salt, submergence, heat and drought stress. Furthermore, one polymorphic site in AiCBLL7 was identified by association analysis which was closely associated with pod length (PL), pod width (PW), hundred pod weight (HPW) and hundred seed weight (HSW). The results of this study provide a foundation for further research on the function of the CBLL gene family in peanut.

Keywords: CBLL gene family; cis-acting element; expression pattern; genome-wide association analysis; peanut.

FIGURE 1

Comparison of the gene structure and motif of 29 CBLL genes in peanut. (A) Unrooted phylogenetic tree with over 50% bootstrap value above the branch. The clade I, II, III, Ⅳ and Ⅴ were displayed in pink, orange, green, blue and dark blue colors, separately. The names of species were abbreviated to two letters, named as Arachis duranensis (Ad), Arachis ipaensis (Ai), Arachis hypogaea L. Tifrunner (Ah). (B) Exon/introns and untranslated regions (UTRs) of CBLLs. Green boxes denote UTR (untranslated region); yellow boxes denote CDS (coding sequence); black lines denote introns. The length of protein can be estimated using the scale at the bottom. (C) Motif architectures of all CBLL genes. Each motif is illustrated with a specific color, and the distribution of identified motifs corresponds to their positions.

FIGURE 2

Chromosomal locations of peanut CBLL genes. Chromosomal positions of the peanut CBLL genes were mapped based on data from PeanutBase. The chromosome number was indicated above each chromosome. Genes in red indicated wild species specific.

FIGURE 3

Chromosomal distribution and gene duplications of the AdCBLL, AiCBLL, and AhCBLL genes. The scales on the circle were in Megabases. Each colored bar represented a chromosome as indicated. Gene IDs were labeled on the basis of their positions on the chromosomes. Red frames indicated wild species specific CBLL genes.

FIGURE 4

Comparative physical mapping showed the degree of orthologous relationships of AhCBLL genes with (A) peanut, (B) Arabidopsis, (C) grape and (D) soybean. Red arrows indicated common collinear CBLL gene pairs.

FIGURE 5

Identification of cis-acting elements in all the peanut CBLL genes. (A) Distribution of cis-acting elements in 2 kb upstream of each AdCBLLs. (B) Distribution of cis-acting elements in 2 kb upstream of each AiCBLLs. (C) Distribution of cis-acting elements in 2 kb upstream of each AhCBLLs. The different colored boxes indicate distinct promoter elements. (D) Assessment AdCBLL different subclass and category proportions in a radar chart. (E) Assessment AiCBLL different subclass and category proportions in a radar chart. (F) Assessment AhCBLL different subclass and category proportions in a radar chart. The lengths of the petals are proportional to the number of elements in each subclass qualitatively. Purple, blue, green and red petals represent plant growth regulation, abiotic stress responses, phytohormone and light responsiveness, respectively.

FIGURE 6

The expression profiles of AhCBLL genes. The heat map of the AhCBLL gene expression levels was hierarchically clustered using the R package “pheatmap” with the data normalization method of Z-score standardization. The color scale bar ranging from blue to red represents low and high expression, respectively. Abbreviation of the 22 tissues used in the expression profiles of AhCBLL genes were as following: seedling leaf 10 days post emergence (leaf 1), main stem leaf (leaf 2), lateral stem leaf (leaf 3), vegetative shoot tip from main stem (veg shoot), reproductive shoot tip from first lateral (repr shoot), 10-day roots (root), 25-day nodules (nodule), perianth, stamen, pistil, aerial gynophore tip (peg tip 1), subterranean peg tip (peg tip 2), Pattee 1 stalk (peg tip Pat. 1), Patte 1 pod (fruit Pat. 1), Pattee 3 pod (fruit Pat.3), Pattee 5 pericarp (pericarp Pat.5), Pattee 6 pericarp (pericarp Pat.6), Pattee 5 seed (seed Pat. 5), Pattee 6 seed (seed Pat. 6), Pattee 7 seed (seed Pat.7), Pattee 8 seed (seed Pat. 8), Pattee 10 seed (seed Pat.10).

FIGURE 7

Relative expression of AhCBLLs under 6-BA, NAA, ACC, GA, MeJA and ABA treatments. Expression characteristics of AhCBLLs in response to different phytohormone at six-time points (00, 01, 03, 06, 09 and 12 h) were normalized to 00 h treatment. The fold changes values were calculated by the 2^−△△Ct method and log2 and represented in color scale legend at the left of the heatmap: red indicated up-regulation and blue showed down-regulated expression.

FIGURE 8

Expression changes of AhCBLLs under different abiotic stresses. The abbreviation represented as following, Sub: submergence, Cold4: cold of 4°C, H40: the heat of 40°C, NaCl: 200 mM NaCl, and PEG, polyethylene glycol.

FIGURE 9

Association mapping results and the phenotypes of the polymorphic sites of the peanut CBLLs associated with HPW/HSW/PL/PW variation. (A) Association results between HPW/HSW/PL/PW and the polymorphisms in peanut CBLLs. (B) Sequences of site significantly associated with HPW/HSW/PL/PW variation. (C) The gene structures of AiCBLL7. (D) Phenotypic comparison of haplotypes of the associated site with HPW/HSW/PL/PW in five environments of the population.