Genomic and transcriptomic comparison of nucleotide variations for insights into bruchid resistance of mungbean (Vigna radiata [L.] R. Wilczek)

Abstract

Background: Mungbean (Vigna radiata [L.] R. Wilczek) is an important legume crop with high nutritional value in South and Southeast Asia. The crop plant is susceptible to a storage pest caused by bruchids (Callosobruchus spp.). Some wild and cultivated mungbean accessions show resistance to bruchids. Genomic and transcriptomic comparison of bruchid-resistant and -susceptible mungbean could reveal bruchid-resistant genes (Br) for this pest and give insights into the bruchid resistance of mungbean.

Results: Flow cytometry showed that the genome size varied by 61 Mb (mega base pairs) among the tested mungbean accessions. Next generation sequencing followed by de novo assembly of the genome of the bruchid-resistant recombinant inbred line 59 (RIL59) revealed more than 42,000 genes. Transcriptomic comparison of bruchid-resistant and -susceptible parental lines and their offspring identified 91 differentially expressed genes (DEGs) classified into 17 major and 74 minor bruchid-resistance-associated genes. We found 408 nucleotide variations (NVs) between bruchid-resistant and -susceptible lines in regions spanning 2 kb (kilo base pairs) of the promoters of 68 DEGs. Furthermore, 282 NVs were identified on exons of 148 sequence-changed-protein genes (SCPs). DEGs and SCPs comprised genes involved in resistant-related, transposable elements (TEs) and conserved metabolic pathways. A large number of these genes were mapped to a region on chromosome 5. Molecular markers designed for variants of putative bruchid-resistance-associated genes were highly diagnostic for the bruchid-resistant genotype.

Conclusions: In addition to identifying bruchid-resistance-associated genes, we found that conserved metabolism and TEs may be modifier factors for bruchid resistance of mungbean. The genome sequence of a bruchid-resistant inbred line, candidate genes and sequence variations in promoter regions and exons putatively conditioning resistance as well as markers detecting these variants could be used for development of bruchid-resistant mungbean varieties.

Fig. 1

Transcriptome analysis of bruchid-resistant–associated genes in mungbean. Bruchid-resistant–associated genes were selected from transcripts per million (TPM) fold change comparison and DESeq analysis of transcriptomes between brucnid-resistant and -susceptible mungbean. The number of DEGs selected by each criterion is indicated. Up and down represent the genes up- and downregulated, respectively, in bruchid-resistant mungbean

Fig. 2

RT-qPCR validation of differentially expressed genes (DEGs). RT-qPCR results of the pattern of gene expression between bruchid-resistant and -susceptible mungbean. The Y axis indicates the relative quantity (RQ) of gene expression with mungbean VrActin (g12676) used as a control. Data are RQ ± SE of ΔΔCT from three experimental repeats. The X axis indicates different bruchid-resistant (R) and -susceptible (S) mungbean lines. Asterisk indicates that the expression of the gene was not detected in the parental line NM92 with CT value set to 40 cycles for calculating the RQ of gene expression

Fig. 3

Pie chart representing the functional categories of DEGs and sequence-changed-protein genes (SCPs). DEGs (a) and SCPs (b) were functionally classified into categories based on annotation and the putative protein domains they harbored. The number of genes in each category is indicated in parentheses

Fig. 4

Validation of nucleotide variations (NVs) identified by RNA sequence comparison. Gene g662 was used to illustrate the verification of NVs. The upper panel shows the cDNA sequence of g662 and the seven NVs (mark in red) identified by RNA sequence comparison of bruchid-resistant (R) and -susceptible (S) mungbean. The NVs in parentheses show the nucleotides in R mungbean (the former letter) changed to that in S mungbean (the latter letter). The lower panel shows the validation of NVs by genomic sequencing between R mungbean lines RIL59 andTC1966 and S line NM92. The color of the letter is synchronized with that of the chromatogram for easy reading. The box indicates the site of NVs. The order of NV sites starts from down-left then down-right panels

Fig. 5

Map of bruchid-resistant–associated genes on chromosome 5 (Vr5) of VC1973A. The corresponding scaffold for each gene in RIL59 is at both sides. The two bruchid-resistant markers are in red. The DEGs are indicated in blue and SCPs in black. The DEGs with an asterisk are also SCPs. For DEGs, the 2-kb promoter sequences were used for mapping, whereas for SCPs, the gene sequences were used

Fig. 6

Close-up map of g39185 and g34480 promoter sequence (a) and g28730 gene (b) on mungbean Vr5. The 2-kb promoter sequences of g39185 (g39185_p) and g34480 (g34480_p) and g27830 gene of RIL59 are strikingly different from that of VC1973A. The number on Vr5 of V1973A indicates the position on the chromosome. mb, million base

Fig. 7

Bruchid-resistant–associated markers of mungbean. Markers designed from promoter sequences g779p, g34480p, and g34458p were used for selecting bruchid-resistant (R) and -susceptible (S) mungbean. The numbers 1 to 9 indicate different mungbean lines, named TC1966, RIL59, NM92, RIL38, RIL39, RIL54, RIL55, RIL153 and RIL156, respectively. PCR products of g779p and g34458p were analyzed on 4 % agarose gel and that of g34480p on 1 % agarose gel