Genome-wide comparative analysis of the nucleotide-binding site-encoding genes in four Ipomoea species

Abstract

The nucleotide-binding site (NBS)-encoding gene is a major type of resistance (R) gene, and its diverse evolutionary patterns were analyzed in different angiosperm lineages. Until now, no comparative studies have been done on the NBS encoding genes in Ipomoea species. In this study, various numbers of NBS-encoding genes were identified across the whole genome of sweet potato (Ipomoea batatas) (#889), Ipomoea trifida (#554), Ipomoea triloba (#571), and Ipomoea nil (#757). Gene analysis showed that the CN-type and N-type were more common than the other types of NBS-encoding genes. The phylogenetic analysis revealed that the NBS-encoding genes formed three monophyletic clades: CNL, TNL, and RNL, which were distinguished by amino acid motifs. The distribution of the NBS-encoding genes among the chromosomes was non-random and uneven; 83.13, 76.71, 90.37, and 86.39% of the genes occurred in clusters in sweet potato, I. trifida, I. triloba, and I. nil, respectively. The duplication pattern analysis reveals the presence of higher segmentally duplicated genes in sweet potatoes than tandemly duplicated ones. The opposite trend was found for the other three species. A total of 201 NBS-encoding orthologous genes were found to form synteny gene pairs between any two of the four Ipomea species, suggesting that each of the synteny gene pairs was derived from a common ancestor. The gene expression patterns were acquired by analyzing using the published datasets. To explore the candidate resistant genes in sweet potato, transcriptome analysis has been carried out using two resistant (JK20 and JK274) and susceptible cultivars (Tengfei and Santiandao) of sweet potato for stem nematodes and Ceratocystis fimbriata pathogen, respectively. A total of 11 differentially expressed genes (DEGs) were found in Tengfei and JK20 for stem nematodes and 19 DEGs in Santiandao and JK274 for C. fimbriata. Moreover, six DEGs were further selected for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis, and the results were consistent with the transcriptome analysis. The results may provide new insights into the evolution of NBS-encoding genes in the Ipomoea genome and contribute to the future molecular breeding of sweet potatoes.

Keywords: Ipomoea species; NBS-encoding gene; chromosomal location; disease resistance; expression analysis; phylogenetic relationship; syntenic analysis.

FIGURE 1

Six conserved motifs in the nucleotide-binding site (NBS) domain of the four Ipomoea species. The amino acids (aa) of the six conserved motifs are extracted. Larger letters indicate higher frequency.

FIGURE 2

Phylogenetic relationships of the nucleotide-binding site (NBS)-encoding genes in Ipomoea batatas, Ipomoea trifida, I. triloba, I. nil, and Arabidopsis based on the amino acids (aa) of conserved NBS domains. Red, cyan-blue, olive, purple, and green lines represent the NBS-encoding genes in I. batatas, I. trifida, I. triloba, I. nil, and Arabidopsis, respectively. P25941 was used as an outgroup (marked as blue). Subclades are named as CNL-A to CNL-D and TNL-A to TNL-H.

FIGURE 3

Distribution of nucleotide-binding site (NBS)-encoding genes across chromosomes. (A) Distribution in sweet potato chromosomes. (B) Distribution in I. trifida chromosomes. (C) Distribution in I. triloba chromosomes. (D) Distribution in I. nil chromosomes. The red color indicates the tandemly duplicated NBS-encoding genes on the chromosomal positions.

FIGURE 4

Collinear gene pairs for I. trifida nucleotide-binding site (NBS) genes on 15 chromosomes of four Ipomoea species: (A) I. batatas; (B) I. trifida; (C) I. triloba; (D) I. nil. The outer circle represents the haploid chromosomes (light pink); the second circle represents the matches of the genes with the genome of (NBS-encoding genes: black; non-NBS-encoding genes: yellow). Red lines show the collinear gene pairs for NBS-encoding genes.

FIGURE 5

Synteny analysis of nucleotide-binding site (NBS)-encoding genes. (A) Synteny between sweet potato and I. trifida. (B) Synteny between sweet potato and I. triloba. (C) Synteny between sweet potato and I. nil. (D) Synteny between I. trifida and I.triloba. (E) Synteny between I. trifida and I. nil. (F) Synteny between I.triloba and I.nil. (G) Schematic representation of syntenic genes among sweet potato, I. trifida, I. triloba, and I. nil. The chromosomes of sweet potato, I. trifida, I. triloba, and I. nil were assigned with red, green, blue, and purple, respectively. Gray lines connect matched gene pairs, with NBS-encoding gene pairs highlighted in pink.

FIGURE 6

Heatmap of the compared expression profiles of duplicated nucleotide-binding site (NBS)-encoding genes in the four Ipomoea species. The heatmap for duplicated NBS-encoding gene pair was named as “gene1” + “^&” + “gene2” + “duplicated patterns (-S: segmental duplication; -T: tandem duplication)” (gene1 and gene2 were duplicated gene pairs). For each tissue or stress: “-1” indicates the heatmap of “gene1”; “-2” indicates the heatmap of “gene2.” (A) Expression profiles of duplicated NBS-encoding genes in different sweet potato tissues: initiative storage root (ISR), distal end (DE), proximal end (PE), root body (RB), and root stalk (RS). (B) Expression profiles of duplicated NBS-encoding genes in different I. trifida tissues: the callus flower, callus stem, flower, flower bud, root 1, root 2, leaf, and stem. (C) Expression profiles of duplicated NBS-encoding genes in different I. triloba tissues: root 1, root 2, flower, flower bud, leaf, and stem. (D) Expression profiles of NBS-encoding genes in different I. nil tissues: the root, embryo, seed coat, flower, leaf, and stem. (E) Expression profiles of sweet potato duplicated NBS-encoding genes in response to various stress: drought, salt, methyl jasmonate (MeJa), abscisic acid (ABA), and salicylic acid (SA). (F) Expression profiles of I. trifida duplicated NBS-encoding genes in response to various stress: beta-aminobutyric acid biotic stress experiment (TTF_BABA), cold stress at 10/4°C day/night experiment (TTF_COLD), biotic stress control (TTF_BICO), benzothiadiazole S-methylester biotic stress experiment (TTF_BTHT), 6-benzylaminopurine 10 uM hormone stress experiment (TTF_BAPT), hormone control experiment (TTF_HOCO), Indole-3-acetic acid 10 uM hormone stress experiment (TTF_IAAT), gibberellic acid 50 uM hormone stress experiment (TTF_GA3T), heat stress at 35/35°C day/night experiment (TTF_HEAT), drought and salt control experiment (TTF_DSCO), heat control at 28/22°C day/night experiment (TTF_HECO), cold control at 28/22°C day/night experiment (TTF_COCO), NaCl salt stress experiment (TTF_NACL), mannitol drought stress experiment (TTF_MANN) and abscisic acid 50 uM hormone stress experiment (TTF_ABAT). (G) Expression profiles of I. triloba duplicated NBS- encoding genes in response to various stress: beta-aminobutyric acid biotic stress experiment (TTB_BABA), cold stress at 10/4°C day/night experiment (TTB_COLD), biotic stress control (TTB_BICO), benzothiadiazole S-methylester biotic stress experiment (TTB_BTHT), 6-benzylaminopurine 10 uM hormone stress experiment (TTB_BAPT), hormone control experiment (TTB_HOCO), Indole-3-acetic acid 10 uM hormone stress experiment (TTB_IAAT), gibberellic acid 50 uM hormone stress experiment (TTB_GA3T), heat stress at 35/35°C day/night experiment (TTB_HEAT), drought and salt control experiment (TTB_DSCO), heat control at 28/22°C day/night experiment (TTB_HECO), cold control at 28/22°C day/night experiment (TTB_COCO), NaCl salt stress experiment (TTB_NACL), mannitol drought stress experiment (TTB_MANN), and abscisic acid 50 uM hormone stress experiment (TTB_ABAT).

FIGURE 7

Heatmap of compared expression profiles of syntenic nucleotide-binding site (NBS)-encoding genes in the four Ipomoea species. The heatmap of syntenic NBS-encoding gene set was named as “sweet potato gene-” + “I. trifida gene-” + “I. triloba gene-” + “I. nil gene.” (A) Heatmap of compared expression profiles of syntenic NBS-encoding genes in the four Ipomoea species tissues. (B) Heatmap of compared expression profiles of syntenic NBS-encoding genes in the four Ipomoea species in response to stress.

FIGURE 8

Heatmap of the expression profiles of differentially expressed genes (DEGs) in response to sweet potato stem nematodes and Ceratocystis fimbriata resistance. (A) DEGs in “Tengfei” and “JK20” under control and sweet potato stem nematodes inoculation. (B) DEGs in “Santiandao” and “JK274” under control and C. fimbriata inoculation. C, control; T, treatment.

FIGURE 9

Expression analysis of nucleotide-binding site (NBS)-encoding genes on the storage roots of sweet potato cultivars or lines. (A) Relative expression levels of g4317.t1, g12493.t1, and g61593.t1 in storage roots after different times of stem nematode infection. (B) Relative expression levels of g4317.t1, g21889.t1, and g8106.t1 after different times of Ceratocystis fimbriata infection. The significance of DEG levels compared with control were denoted as ^∗<0.05, ^∗∗<0.01.