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This paper reports fine mapping of qCLS for resistance to Cercospora leaf spot disease in mungbean and identified LOC106765332encoding TATA-binding-protein-associated factor 5 (TAF5) as the candidate gene for the resistance Cercospora leaf spot (CLS) caused by the fungus Cercospora canescens is an important disease of mungbean. A QTL mapping using mungbean F2 and BC1F1 populations developed from the “V4718” (resistant) and “Kamphaeng Saen 1” (KPS1; susceptible) has identified a major QTL controlling CLS resistance (qCLS). In this study, we finely mapped the qCLS and identified candidate genes at this locus. A BC8F2 [KPS1 × (KPS1 × V4718)] population developed in this study and the F2 (KPS1 × V4718) population used in a previous study were genotyped with 16 newly developed SSR markers. QTL analysis in the BC8F2 and F2 populations consistently showed that the qCLS was mapped to a genomic region of ~ 13 Kb on chromosome 6, which contains only one annotated gene, LOC106765332 (designated “VrTAF5”), encoding TATA-binding-protein-associated factor 5 (TAF5), a subunit of transcription initiation factor IID and Spt-Ada-Gcn5 acetyltransferase complexes. Sequence comparison of VrTAF5 between KPS1 and V4718 revealed many single nucleotide polymorphisms (SNPs) and inserts/deletions (InDels) in which eight SNPs presented in eight different exons, and an SNP (G4,932C) residing in exon 8 causes amino acid change (S250T) in V4718. An InDel marker was developed to detect a 24-bp InDel polymorphism in VrTAF5 between KPS1 and V4718. Analysis by RT-qPCR showed that expression levels of VrTAF5 in KPS1 and V4718 were not statistically different. These results indicated that mutation in VrTAF5 causing an amino acid change in the VrTAF5 protein is responsible for CLS resistance in V4718.

Black gram (Vigna mungo var. mungo) is an important pulse crop in Asia. The cowpea weevil (Callosobruchus maculatus) is a stored-seed insect pest (seed weevil/bruchid) that causes serious postharvest losses in pulse crops, including black gram. In this study, we constructed a high-density linkage map for black gram and identified quantitative trait loci (QTLs) for C. maculatus resistance. A recombinant inbred line (RIL) population of 150 lines from a cross between BC48 [cultivated black gram (var. mungo); bruchid-susceptible] and TC2210 [wild black gram (var. silvestris); bruchid-resistant] were used to construct a linkage map of 3,675 SNP markers from specific-locus amplified fragment sequencing. The map comprised 11 linkage groups spanning 1,588.7 cM with an average distance between adjacent markers of 0.57 cM. Seeds of the RIL population grown in 2016 and 2017 were evaluated for C. maculatus resistance through two traits; the percentage of damaged seeds (PDS) and infestation severity progress (AUDPS). Inclusive composite interval mapping identified three QTLs each for PDS and AUDPS. Two QTLs, qVmunBr6.1 and qVmunBr6.2, mapped about 10 cM apart on linkage group 6 were common between PDS and AUDPS. Comparative genome analysis revealed that qVmunBr6.1 and qVmunBr6.2 are new loci for C. maculatus resistance in Vigna species and that genes encoding a lectin receptor kinase and chitinase are candidates for qVmunBr6.2. The high-density linkage map constructed and QTLs for bruchid resistance identified in this study will be useful for molecular breeding of black gram.

The moth bean (Vigna aconitifolia), possibly the most primitive crop of the genus Vigna, is a highly drought- and heat-resistant legume grown in arid areas. Moth bean domestication involved phenotypic changes, including reduction of seed dormancy and pod shattering, increased organ size, and earlier flowering and maturity. However, the genetics of the domestication process in moth bean is not known. In this study, we constructed a genetic linkage map for moth bean and used the map to identify quantitative trait loci (QTL) for domestication-related traits of an F2 population of 188 individuals produced from a cross of wild moth bean (TN67) and cultivated moth bean (ICPMO056). The genetic linkage map comprised 11 linkage groups (LG) of 172 simple sequence repeat markers and spanned a total length of 1016.8 centiMorgan (cM), with an average marker distance of 7.34 cM. A comparative genome analysis showed high genome synteny between moth bean and mungbean (Vigna radiata), adzuki bean (Vigna angularis), rice bean (Vigna umbellata), and yardlong bean (Vigna unguiculata). In total, 50 QTLs and 3 genes associated with 20 domestication-related traits were identified. Most of the QTLs belonged to five LGs (1, 2, 4, 7, and 10). Key traits related to domestication such as seed dormancy and pod shattering were controlled by large-effect QTLs (PVE > 20%) with one or two minor QTLs, whereas all other traits were controlled by one–seven minor QTLs, apart from seed weight, which was controlled by one major and seven minor QTLs. These results suggest that a small number of mutations with large phenotypic effects have contributed to the domestication of the moth bean. Comparative analysis of QTLs with related Vigna crops revealed that there are several domestication-related large-effect QTLs that had not been used in moth bean domestication. This study provides a basic genetic map and identified genome regions associated with domestication-related traits, which will be useful for the genetic improvement of the moth bean and related Vigna species.

Luffa acutangula and Luffa aegyptiaca are domesticated plants in the family Cucurbitaceae. They are mainly cultivated in the tropical and subtropical regions of Asia. The chloroplast genomes of many Cucurbitaceae species were sequenced to examine gene content and evolution. However, the chloroplast genome sequences of L. acutangula and L. aegyptiaca have not been reported. We report the first complete sequences of L. acutangula and L. aegyptiaca chloroplast genomes obtained from Pacific Biosciences sequencing and use them to infer evolutionary relationships. The chloroplast genomes of L. acutangula and L. aegyptiaca are 157,202 and 157,275 bp, respectively. Both genomes possessed the typical quadripartite structure and contained 131 genes, including 87 coding genes, 36 tRNA genes and 8 rRNA genes. We identified simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP) from both chloroplast genomes. Polycistronic mRNA was examined in L. acutangula and L. aegyptiaca using RNA sequences from Isoform sequencing to identify co-transcribed genes. IR size and locations were compared to other species and found to be relatively unchanged. Phylogenetic analysis confirmed the close relationship between L. acutangula and L. aegyptiaca in the Cucurbitaceae lineage and showed separation of the Luffa monophyletic clade from other species in the subtribe Sicyocae. The results obtained from this study can be useful for studying the evolution of Cucurbitaceae plants.

Vigna mungo is cultivated in approximately 5 million hectares worldwide. The chloroplast genome of this species has not been previously reported. In this study, we sequenced the genome and transcriptome of the V. mungo chloroplast. We identified many positively selected genes in the photosynthetic pathway (e.g., rbcL, ndhF, and atpF) and RNA polymerase genes (e.g., rpoC2) from the comparison of the chloroplast genome of V. mungo, temperate legume species, and tropical legume species. Our transcriptome data from PacBio isoform sequencing showed that the 51-kb DNA inversion could affect the transcriptional regulation of accD polycistronic. Using Illumina deep RNA sequencing, we found RNA editing of clpP in the leaf, shoot, flower, fruit, and root tissues of V. mungo. We also found three G-to-A RNA editing events that change guanine to adenine in the transcripts transcribed from the adenine-rich regions of the ycf4 gene. The edited guanine bases were found particularly in the chloroplast genome of the Vigna species. These G-to-A RNA editing events were likely to provide a mechanism for correcting DNA base mutations. The V. mungo chloroplast genome sequence and the analysis results obtained in this study can apply to phylogenetic studies and chloroplast genome engineering.

Loss/reduction of function of Mildew Locus O (MLO) genes clade V and MLO clade IV has been shown to be responsible for powdery mildew (PM) resistance in several plant species. Mungbean (Vigna radiata) genome possesses 18 MLO genes, VrMLO1 - VrMLO18. A previous study using mungbean F2 and BC1F1 populations derived from a cross between "CN60″ (susceptible) and "RUM5″ (resistance) demonstrated that QTL qPMRUM5-3 is a major QTL for PM resistance caused by Erysiphe polygoni and is the same with major QTL qPMV4718-3 that confers PM resistance in "V4718″ (resistance). In this study, bioinformatics analysis revealed VrMLO12 locates in the qPMRUM5-3 region. Fine mapping in the F2 and BC1F1 populations using newly developed DNA markers including gene-specific markers demonstrated association between VrMLO12 and the PM resistance. Sequence analyses of VrMLO12 revealed that compared to susceptible mungbeans, RUM5 and V4718 possess SNPs in exon 10 and exon 13. The SNPs caused amino acid changes of VrMLO12, A387S and A476 G, respectively. The change occurred in transmembrane 6 domain and calmodulin binding domain (CaMBD) of the VrMLO12 protein, respectively. qRT-PCR showed that transcript expression level of VrMLO12 in RUM5 challenged with and without by E. polygoni was significantly higher than that in CN60. Phylogenetic analysis revealed that in contrast to previous findings that MLO proteins associated with PM resistance belong to MLO clade V and MLO clade IV, VrMLO12 belongs to MLO clade II. The result suggested that VrMLO12 may function differently from the other MLOs that associated with PM susceptibility. Our findings provide insight into the PM resistance in mungbean and tools for mungbean breeding.

The azuki bean weevil (Callosobruchus chinensis L.) is an insect pest responsible for serious postharvest seed loss in leguminous crops. In this study, we performed quantitative trait locus (QTL) mapping of seed resistance to C. chinensis in moth bean (Vigna aconitifolia [Jaqc.] Maréchal). An F2 population of 188 plants developed by crossing resistant accession ‘TN67’ (wild type from India, male parent) and susceptible accession ‘IPCMO056’ (cultivated type from India, female parent) was used for mapping. Seeds of the F2 population from 2014 and F2:3 populations from 2016 and 2017 were bioassayed with C. chinensis, and the percentage of damaged seeds and progress of infestation severity were measured. Segregation analysis suggested that C. chinensis resistance in TN176 is controlled by a single dominant gene, designated as Rcc. QTL analysis revealed one principal and one modifying QTL for the resistance, named qVacBrc2.1 and qVacBrc5.1, respectively. qVacBrc2.1 was located on linkage group 2 between simple sequence repeat markers CEDG261 and DMB-SSR160 and accounted for 50.41% to 64.23% of resistance-related traits, depending on the trait and population. Comparative genomic analysis suggested that qVacBrc2.1 is the same as QTL Brc2.1 conferring C. chinensis resistance in wild azuki bean (V. nepalensis Tateishi and Maxted). Markers CEDG261 and DMB-SSR160 should be useful for marker-assisted selection for C. chinensis resistance in moth bean.

Sequence analysis and genetic mapping revealed that a 1,444 bp deletion causes a premature stop codon in SbBADH2 of sorghum IS19912. The non-function of SbBADH2 is responsible for fragrance in sorghum IS19912. 2-acetyl-1-pyrroline (2AP) is a potent volatile compound causing fragrance in several plants and foods. Seeds of some varieties of rice, sorghum and soybean possess fragrance. The genes responsible for fragrance in rice and soybean are orthologs that correspond to betaine aldehyde dehydrogenase 2 (BADH2). Genotypes harboring fragrance in rice and soybean contain a premature stop codon in BADH2 which impairs the synthesis of full length functional BADH2 protein leading to the accumulation of 2AP. In this study, we reported an association between the BADH2 gene and fragrance in sorghum. An F2 population of 187 plants developed from a cross between KU630 (non-fragrant) and IS19912 (fragrant) was used. Leaves of F2 and F3 progenies were evaluated for fragrance by organoleptic test, while seeds of F2 plants were analyzed for 2AP. The tests consistently showed that the fragrance is controlled by a single recessive gene. Gene expression analysis of SbBADH1 and SbBADH2 in leaves of KU630 and IS19912 at various stages revealed that SbBADH1 and SbBADH2 were expressed in both accessions. Sequence comparison between KU630 and IS19912 revealed a continuous 1,444 bp deletion encompassing exon 12 to 15 of SbBADH2 in IS19912 which introduces a frameshift mutation and thus causes a premature stop codon. An indel marker was developed to detect polymorphism in SbBADH2. Bulk segregant and QTL analyses confirmed the association between SbBADH2 and fragrance.

Sequence analysis revealed that an SNP (A1855G) in CsBADH of cucumber accession PK2011T202 causes amino acid change in a highly conserved motif, Y163C. Gene mapping showed association between the SNP and the fragrance. Pandan-like fragrance is a value-added trait in several food crops such as rice, vegetable soybean and sorghum. The fragrance is caused by the volatile chemical 2-acetyl-1-pyrroline (2AP). Mutation(s) in betaine aldehyde dehydrogenase 2 (BADH2; also known as aminoaldehyde dehydrogenase 2) gene causes defective BADH2 and results in biosynthesis of 2AP. Recently, cucumber cultivars possessing pandan-like fragrance were discovered in Thailand. In this study, we report an association between CsBADH and the fragrance in cucumber accession "PK2011T202". Gene expression analysis of CsBADH in leaves of PK2011T202 and "301176" (non-fragrant) at various growth stages revealed that CsBADH was expressed in both accessions. Sequence comparison of CsBADH showed that PK2011T202 possesses a single base substitution (A1855G) in exon 5 which causes an amino acid change in a highly conserved motif of BADH, Y163C. Single nucleotide-amplified polymorphism markers were developed to detect the SNP polymorphism between the wild-type and fragrance alleles. Since CsBADH is located on chromosome 1, quantitative trait locus (QTL) mapping was conducted for this chromosome using an F2 and a backcross populations developed from the cross between PK2011T202 and 301176. QTL analysis in both populations showed that the major QTL for fragrance, qFgr, was co-localized with the CsBADH. We concluded that the defect function of CsBADH is responsible for fragrance in cucumber PK2011T202.