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Expression patterns, molecular markers and genetic diversity of insect-susceptible and resistant Barbarea genotypes by comparative transcriptome analysis.

Zhang X, Liu T, Wei X, Qiu Y, Song J, Wang H, Shen D, Agerbirk N, Li X - BMC Genomics (2015)

Bottom Line: However, gene expression for two downstream enzymes, the glucosyl transferase (UGT73C11) and an oxidosqualene cyclase (OSC), were significantly upregulated in the P-type compared with the G-type plant.The homologous genes from P- and G-type plants were detected by BLAST unigenes with a cutoff level E-value < e(-10). 12,980 gene families containing 26,793 P-type and 36,944 G-type unigenes were shared by the two types of B. vulgaris. 38,397 single nucleotide polymorphisms (SNPs) were found in 9,452 orthologous genes between the P- and G-type plants.These data represent useful information for pest-resistance gene mining and for the investigation of the molecular basis of plant-pest interactions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences; Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing, 100081, China. zhangxiaohui01@caas.cn.

ABSTRACT

Background: Barbarea vulgaris contains two genotypes: the glabrous type (G-type), which confers resistance to the diamondback moth (DBM) and other insect pests, and the pubescent type (P-type), which is susceptible to the DBM. Herein, the transcriptomes of P-type B. vulgaris before and after DBM infestation were subjected to Illumina (Solexa) pyrosequencing and comparative analysis.

Results: 5.0 gigabase pairs of clean nucleotides were generated. Non-redundant unigenes (33,721) were assembled and 94.1 % of them were annotated. Compared with our previous G-type transcriptome, the expression patterns of many insect responsive genes, including those related to secondary metabolism, phytohormones and transcription factors, which were significantly induced by DBM in G-type plants, were less sensitive to DBM infestation in P-type plants. The genes of the triterpenoid saponin pathway were identified in both G- and P-type plants. The upstream genes of the pathway showed similar expression patterns between the two genotypes. However, gene expression for two downstream enzymes, the glucosyl transferase (UGT73C11) and an oxidosqualene cyclase (OSC), were significantly upregulated in the P-type compared with the G-type plant. The homologous genes from P- and G-type plants were detected by BLAST unigenes with a cutoff level E-value < e(-10). 12,980 gene families containing 26,793 P-type and 36,944 G-type unigenes were shared by the two types of B. vulgaris. 38,397 single nucleotide polymorphisms (SNPs) were found in 9,452 orthologous genes between the P- and G-type plants. We also detected 5,105 simple sequence repeats (SSRs) in the B. vulgaris transcriptome, comprising mono-nucleotide-repeats (2,477; 48.5 %) and triple-nucleotide-repeats (1,590; 31.1 %). Of these, 1,657 SSRs displayed polymorphisms between the P- and G-type. Consequently, 913 SSR primer pairs were designed with a resolution of more than two nucleotides. We randomly chose 30 SSRs to detect the genetic diversity of 32 Barbarea germplasms. The distance tree showed that these accessions were clearly divided into groups, with the G-type grouping with available Western and Central European B. vulgaris accessions in contrast to the P-type accession, B. stricta and B. verna.

Conclusions: These data represent useful information for pest-resistance gene mining and for the investigation of the molecular basis of plant-pest interactions.

No MeSH data available.


Related in: MedlinePlus

Comparative transcription profiles of the putative genes in the triterpene saponin synthetic pathway in P- and G-type B. vulgaris. a, Heatmaps representing the expression levels of different genes/families determined by RNA-seq. The color bar is shown on the top right. Data represent log (RPKM) of each treatment. The metabolites of the pathway are shown in bright green on top and the enzymes for each metabolic step are shown below in black using the following abbreviations: MVA, mevalonic acid; MEP, 2-C-methyl-D-erythritol 4-phosphate; IPP, isopentenyl diphosphate; G3P, glyceraldehyde 3-phosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; 2,3-OS, 2,3-oxidosqualene; AACT, acetyl-CoA acyltransferase; HMGS, 3-hydroxy-3-methylglutaryl CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; MK, mevalonic acid kinase; PMK, phosphomevalonate kinase; MDD, mevalonic acid diphosphate decarboxylase; DXS, deoxy-D-xylulose 5-phosphate synthase; DXR, deoxy-D-xylulose 5-phosphate reductase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) synthase; HDR, HMB-PP reductase; IDI, IPI isomerase; GPS, geranylgeranyl pyrophosphate synthase; FPS, farnesyl pyrophosphate synthase; SS, squalene synthase; SE, squalene epoxidase; OSC, oxidosqualene cyclases; P450, cytochrome P-450; UGT, UDP-dependent glycosyl transferases. Two stars indicate the two significantly induced genes in P-type plants. b, Q-PCR analysis of three selected genes of the pathway. The ordinates show the relative expression levels; the abscissas show the time points. Error bars indicate the SD of three biological replicates
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Fig3: Comparative transcription profiles of the putative genes in the triterpene saponin synthetic pathway in P- and G-type B. vulgaris. a, Heatmaps representing the expression levels of different genes/families determined by RNA-seq. The color bar is shown on the top right. Data represent log (RPKM) of each treatment. The metabolites of the pathway are shown in bright green on top and the enzymes for each metabolic step are shown below in black using the following abbreviations: MVA, mevalonic acid; MEP, 2-C-methyl-D-erythritol 4-phosphate; IPP, isopentenyl diphosphate; G3P, glyceraldehyde 3-phosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; 2,3-OS, 2,3-oxidosqualene; AACT, acetyl-CoA acyltransferase; HMGS, 3-hydroxy-3-methylglutaryl CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; MK, mevalonic acid kinase; PMK, phosphomevalonate kinase; MDD, mevalonic acid diphosphate decarboxylase; DXS, deoxy-D-xylulose 5-phosphate synthase; DXR, deoxy-D-xylulose 5-phosphate reductase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) synthase; HDR, HMB-PP reductase; IDI, IPI isomerase; GPS, geranylgeranyl pyrophosphate synthase; FPS, farnesyl pyrophosphate synthase; SS, squalene synthase; SE, squalene epoxidase; OSC, oxidosqualene cyclases; P450, cytochrome P-450; UGT, UDP-dependent glycosyl transferases. Two stars indicate the two significantly induced genes in P-type plants. b, Q-PCR analysis of three selected genes of the pathway. The ordinates show the relative expression levels; the abscissas show the time points. Error bars indicate the SD of three biological replicates

Mentions: The genes of the triterpenoid saponin pathway (except P450s) were identified in both G- and P-type B. vulgaris transcriptomes, based on gene annotation. To limit the omissions, the saponin pathway genes were BLAST searched against the transcriptome databases from both G- and P-type plants. Seventy-one G-type and 44 P-type unigenes representing 22 enzymes catalyzing 20 metabolic reactions of the triterpenoid saponin pathway were identified (Additional file 1: Table S10). The presence of more unigenes in the G-type plants could partially reflect the higher heterozygosity among the sequenced individuals; polymorphisms within the alleles could produce multiple potential unigenes during the assembly process. Particularly evident were MDD and LUP2, which were represented by seven CL278.Contigs and five CL2531.Contigs, respectively (Additional file 1: Table S10). The authenticity of these sequences requires further experimental analysis. The expression abundances of these saponin synthesis genes were compared between G- and P-type plants (Fig. 3a and Additional file 1: Table S10); the genes upstream of SE showed similar expression patterns between the two genotypes. Unexpectedly, expression of genes for the bottom first and third enzymes, the glucosyl transferase (UGT73C11) and an oxidosqualene cyclases (OCS), was significantly upregulated, and these mRNAs accumulated by more than 10-fold in the P-type compared with the G-type plant. The over expression of these genes were also comfirmed by Q-PCR analysis, though the change levels were not as high as RNA-Seq displayed (Fig. 3b). One of them, UGT73C11, is known to be functional in the P-type plant [23]. The dramatic induction of these two enzymes could result from the P-type plants suffering more serious damage under insect infestation and because the regulator of this pathway is still functioning. However, some of the enzymes upstream of UGT73C11, most likely the uncharacterized enzyme-P450s, are perhaps dysfunctional or have gained new functions, resulting in no anti-DBM saponin being produced in P-type plants.Fig. 3


Expression patterns, molecular markers and genetic diversity of insect-susceptible and resistant Barbarea genotypes by comparative transcriptome analysis.

Zhang X, Liu T, Wei X, Qiu Y, Song J, Wang H, Shen D, Agerbirk N, Li X - BMC Genomics (2015)

Comparative transcription profiles of the putative genes in the triterpene saponin synthetic pathway in P- and G-type B. vulgaris. a, Heatmaps representing the expression levels of different genes/families determined by RNA-seq. The color bar is shown on the top right. Data represent log (RPKM) of each treatment. The metabolites of the pathway are shown in bright green on top and the enzymes for each metabolic step are shown below in black using the following abbreviations: MVA, mevalonic acid; MEP, 2-C-methyl-D-erythritol 4-phosphate; IPP, isopentenyl diphosphate; G3P, glyceraldehyde 3-phosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; 2,3-OS, 2,3-oxidosqualene; AACT, acetyl-CoA acyltransferase; HMGS, 3-hydroxy-3-methylglutaryl CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; MK, mevalonic acid kinase; PMK, phosphomevalonate kinase; MDD, mevalonic acid diphosphate decarboxylase; DXS, deoxy-D-xylulose 5-phosphate synthase; DXR, deoxy-D-xylulose 5-phosphate reductase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) synthase; HDR, HMB-PP reductase; IDI, IPI isomerase; GPS, geranylgeranyl pyrophosphate synthase; FPS, farnesyl pyrophosphate synthase; SS, squalene synthase; SE, squalene epoxidase; OSC, oxidosqualene cyclases; P450, cytochrome P-450; UGT, UDP-dependent glycosyl transferases. Two stars indicate the two significantly induced genes in P-type plants. b, Q-PCR analysis of three selected genes of the pathway. The ordinates show the relative expression levels; the abscissas show the time points. Error bars indicate the SD of three biological replicates
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Related In: Results  -  Collection

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Fig3: Comparative transcription profiles of the putative genes in the triterpene saponin synthetic pathway in P- and G-type B. vulgaris. a, Heatmaps representing the expression levels of different genes/families determined by RNA-seq. The color bar is shown on the top right. Data represent log (RPKM) of each treatment. The metabolites of the pathway are shown in bright green on top and the enzymes for each metabolic step are shown below in black using the following abbreviations: MVA, mevalonic acid; MEP, 2-C-methyl-D-erythritol 4-phosphate; IPP, isopentenyl diphosphate; G3P, glyceraldehyde 3-phosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; 2,3-OS, 2,3-oxidosqualene; AACT, acetyl-CoA acyltransferase; HMGS, 3-hydroxy-3-methylglutaryl CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; MK, mevalonic acid kinase; PMK, phosphomevalonate kinase; MDD, mevalonic acid diphosphate decarboxylase; DXS, deoxy-D-xylulose 5-phosphate synthase; DXR, deoxy-D-xylulose 5-phosphate reductase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) synthase; HDR, HMB-PP reductase; IDI, IPI isomerase; GPS, geranylgeranyl pyrophosphate synthase; FPS, farnesyl pyrophosphate synthase; SS, squalene synthase; SE, squalene epoxidase; OSC, oxidosqualene cyclases; P450, cytochrome P-450; UGT, UDP-dependent glycosyl transferases. Two stars indicate the two significantly induced genes in P-type plants. b, Q-PCR analysis of three selected genes of the pathway. The ordinates show the relative expression levels; the abscissas show the time points. Error bars indicate the SD of three biological replicates
Mentions: The genes of the triterpenoid saponin pathway (except P450s) were identified in both G- and P-type B. vulgaris transcriptomes, based on gene annotation. To limit the omissions, the saponin pathway genes were BLAST searched against the transcriptome databases from both G- and P-type plants. Seventy-one G-type and 44 P-type unigenes representing 22 enzymes catalyzing 20 metabolic reactions of the triterpenoid saponin pathway were identified (Additional file 1: Table S10). The presence of more unigenes in the G-type plants could partially reflect the higher heterozygosity among the sequenced individuals; polymorphisms within the alleles could produce multiple potential unigenes during the assembly process. Particularly evident were MDD and LUP2, which were represented by seven CL278.Contigs and five CL2531.Contigs, respectively (Additional file 1: Table S10). The authenticity of these sequences requires further experimental analysis. The expression abundances of these saponin synthesis genes were compared between G- and P-type plants (Fig. 3a and Additional file 1: Table S10); the genes upstream of SE showed similar expression patterns between the two genotypes. Unexpectedly, expression of genes for the bottom first and third enzymes, the glucosyl transferase (UGT73C11) and an oxidosqualene cyclases (OCS), was significantly upregulated, and these mRNAs accumulated by more than 10-fold in the P-type compared with the G-type plant. The over expression of these genes were also comfirmed by Q-PCR analysis, though the change levels were not as high as RNA-Seq displayed (Fig. 3b). One of them, UGT73C11, is known to be functional in the P-type plant [23]. The dramatic induction of these two enzymes could result from the P-type plants suffering more serious damage under insect infestation and because the regulator of this pathway is still functioning. However, some of the enzymes upstream of UGT73C11, most likely the uncharacterized enzyme-P450s, are perhaps dysfunctional or have gained new functions, resulting in no anti-DBM saponin being produced in P-type plants.Fig. 3

Bottom Line: However, gene expression for two downstream enzymes, the glucosyl transferase (UGT73C11) and an oxidosqualene cyclase (OSC), were significantly upregulated in the P-type compared with the G-type plant.The homologous genes from P- and G-type plants were detected by BLAST unigenes with a cutoff level E-value < e(-10). 12,980 gene families containing 26,793 P-type and 36,944 G-type unigenes were shared by the two types of B. vulgaris. 38,397 single nucleotide polymorphisms (SNPs) were found in 9,452 orthologous genes between the P- and G-type plants.These data represent useful information for pest-resistance gene mining and for the investigation of the molecular basis of plant-pest interactions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences; Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing, 100081, China. zhangxiaohui01@caas.cn.

ABSTRACT

Background: Barbarea vulgaris contains two genotypes: the glabrous type (G-type), which confers resistance to the diamondback moth (DBM) and other insect pests, and the pubescent type (P-type), which is susceptible to the DBM. Herein, the transcriptomes of P-type B. vulgaris before and after DBM infestation were subjected to Illumina (Solexa) pyrosequencing and comparative analysis.

Results: 5.0 gigabase pairs of clean nucleotides were generated. Non-redundant unigenes (33,721) were assembled and 94.1 % of them were annotated. Compared with our previous G-type transcriptome, the expression patterns of many insect responsive genes, including those related to secondary metabolism, phytohormones and transcription factors, which were significantly induced by DBM in G-type plants, were less sensitive to DBM infestation in P-type plants. The genes of the triterpenoid saponin pathway were identified in both G- and P-type plants. The upstream genes of the pathway showed similar expression patterns between the two genotypes. However, gene expression for two downstream enzymes, the glucosyl transferase (UGT73C11) and an oxidosqualene cyclase (OSC), were significantly upregulated in the P-type compared with the G-type plant. The homologous genes from P- and G-type plants were detected by BLAST unigenes with a cutoff level E-value < e(-10). 12,980 gene families containing 26,793 P-type and 36,944 G-type unigenes were shared by the two types of B. vulgaris. 38,397 single nucleotide polymorphisms (SNPs) were found in 9,452 orthologous genes between the P- and G-type plants. We also detected 5,105 simple sequence repeats (SSRs) in the B. vulgaris transcriptome, comprising mono-nucleotide-repeats (2,477; 48.5 %) and triple-nucleotide-repeats (1,590; 31.1 %). Of these, 1,657 SSRs displayed polymorphisms between the P- and G-type. Consequently, 913 SSR primer pairs were designed with a resolution of more than two nucleotides. We randomly chose 30 SSRs to detect the genetic diversity of 32 Barbarea germplasms. The distance tree showed that these accessions were clearly divided into groups, with the G-type grouping with available Western and Central European B. vulgaris accessions in contrast to the P-type accession, B. stricta and B. verna.

Conclusions: These data represent useful information for pest-resistance gene mining and for the investigation of the molecular basis of plant-pest interactions.

No MeSH data available.


Related in: MedlinePlus