Two genes encoding caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1) are candidate genes for physical seed dormancy in cowpea (Vigna unguiculata (L.) Walp.).

KEY MESSAGE: The major QTL Sdp1.1+ controlling seed dormancy in cowpea was finely mapped, and two CCoAOMT1 genes were identified as candidate genes for the dormancy. Seed dormancy in wild cowpea may be useful in breeding cultivated cowpea with pre-harvest sprouting resistance. A previous study identified a major quantitative trait locus (QTL) for seed dormancy, Sdp1.1+ , using the population of the cross between cultivated cowpea 'JP81610' and wild cowpea 'JP89083.' However, the molecular basis of seed dormancy in cowpea is not yet known. In this study, we aimed to finely map the locus Sdp1.1+ and identify candidate gene(s) for it. Germination tests demonstrated that the seed coat is the major factor controlling seed dormancy in the wild cowpea JP89083. Microscopic observations revealed that wild cowpea seeds, unlike cultivated cowpea seeds, possessed a palisade cuticle layer. Fine mapping using a large F2 population of the cross JP81610 × JP89083 grown in Thailand revealed a single QTL, Sdp1.1+ , controlling seed dormancy. The Sdp1.1+ was confirmed using a small F2 population of the same cross grown in Japan. The Sdp1.1+ was mapped to a 37.34-Kb region containing three genes. Two closely linked genes, Vigun03g278900 (VuCCoAOMT1a) and Vigun03g290000 (VuCCoAOMT1b), located 4.844 Kb apart were considered as candidate genes for seed dormancy. The two genes encoded caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1). DNA sequencing and alignment of VuCCoAOMT1a and VuCCoAOMT1b between JP89083 and JP81610 revealed a single nucleotide polymorphism (SNP) causing an amino acid change in VuCCoAOMT1a and several SNPs leading to six amino acid changes in VuCCoAOMT1b. Altogether, these results indicate that VuCCoAOMT1a and VuCCoAOMT1b are candidate genes controlling physical seed dormancy in the wild cowpea JP89083.

Fine mapping of QTL conferring Cercospora leaf spot disease resistance in mungbean revealed TAF5 as candidate gene for the resistance.

KEY MESSAGE: 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.

Genetic diversity and structure of landrace of lablab (Lablab purpureus (L.) Sweet) cultivars in Thailand revealed by SSR markers.

Lablab (Lablab purpureus (L.) Sweet) is a legume crop widely cultivated in tropical and subtropical regions of Africa and Asia. In this study, we assessed genetic diversity and population structure of 299 individuals of subspecies purpureus and bengalensis of lablab from Thailand using 13 simple sequence repeat (SSR) markers. The SSR markers detected only 34 alleles in total with a mean of 2.6 alleles per locus. Overall gene diversity was 0.360. Gene diversity (H E) and allelic richness (A R) in different geographic regions was comparable. Similarly, both H E and A R between subspecies purpureus and bengalensis were similar. STRUCTURE and neighbor-joining (NJ) analyses revealed that the 299 individuals were clustered into two major groups. In contrast, principal coordinate analysis (PCoA) revealed admixture of the lablab germplasm. STRUCTURE, NJ and PCoA analyses also revealed that the subspecies purpureus and bengalensis are not genetically differentiated. Although the number of individuals from the west of Thailand was small and all of them were collected from the same province, they possessed comparable gene diversity with those from the other geographic regions. These results demonstrated that there is moderately low genetic diversity of lablab in Thailand and the west of the country possesses high diversity of lablab.

A second VrPGIP1 allele is associated with bruchid resistance (Callosobruchus spp.) in wild mungbean (Vigna radiata var. sublobata) accession ACC41.

Two bruchid species, azuki bean weevil (Callosobruchus chinensis L.) and cowpea weevil (Callosobruchus maculatus F.), are the most important insect pests of mungbean [Vigna radiata (L.) Wilczek] after harvest. Improving bruchid resistance is a major goal for mungbean breeders. Bruchid resistance in mungbean is controlled by a single major locus, Br. The tightly linked VrPGIP1 and VrPGIP2, which encode polygalacturonase-inhibiting proteins (PGIPs), are the candidate genes at the Br locus associated with bruchid resistance. One VrPGIP1 resistance allele and two VrPGIP2 resistance alleles have been identified. In this study, we fine-mapped the bruchid-resistance genes in wild mungbean (V. radiata var. sublobata) accession ACC41 using the F2 population (574 individuals) derived from the 'Kamphaeng Saen 2' (susceptible) × ACC41 (resistant) cross. A QTL analysis indicated that the resistance to the azuki bean weevil and cowpea weevil in ACC41 is controlled by a major QTL (qBr5.1) and a minor QTL (qBr5.2), which are only 0.3 cM apart. qBr5.1 and qBr5.2 accounted for about 82% and 2% of the resistance variation in the F2 population, respectively. qBr5.1 was mapped to a 237.35-kb region on mungbean chromosome 5 containing eight annotated genes, including VrPGIP1 and VrPGIP2. An examination of the ACC41 VrPGIP1 and VrPGIP2 sequences revealed a new allele for VrPGIP1 (i.e., VrPGIP1-2). Compared with the wild-type sequence, VrPGIP1-2 has five SNPs, of which four cause amino acid changes (residues 125, 129, 188, and 336). A protein sequence analysis indicated that residues 125 and 129 in VrPGIP1-2 are in a ß-sheet B1 region, whereas residues 188 and 336 are in a C10-helix region and at the end of the C-terminal region, respectively. Because the ß-sheet B1 region is important for interactions with polygalacturonase (PG), residues 125 and 129 in VrPGIP1-2 likely contribute to bruchid resistance by inhibiting PG. Our results imply that VrPGIP1-2 is associated with the bruchid resistance of wild mungbean accession ACC41. This new resistance allele may be useful for breeding mungbean varieties exhibiting durable bruchid resistance.

Two tightly linked genes coding for NAD-dependent malic enzyme and dynamin-related protein are associated with resistance to Cercospora leaf spot disease in cowpea (Vigna unguiculata (L.) Walp.).

Cercospora leaf spot (CLS) caused by Cercospora canescens is an important disease of cowpea (Vigna unguiculata). A previous study using an F2 population [CSR12906 (susceptible) × IT90K-59-120 (resistant)] identified a major QTL qCLS9.1 for resistance to CLS. In this study, we finely mapped and identified candidate genes of qCLS9.1 using an F3:4 population of 699 individuals derived from two F2:3 individuals segregating at qCLS9.1 from the original population. Fine mapping narrowed down the qCLS9.1 for the resistance to a 60.6-Kb region on cowpea chromosome 10. There were two annotated genes in the 60.6-Kb region; Vigun10g019300 coding for NAD-dependent malic enzyme 1 (NAD-ME1) and Vigun10g019400 coding for dynamin-related protein 1C (DRP1C). DNA sequence analysis revealed 12 and 2 single nucleotide polymorphisms (SNPs) in the coding sequence (CDS) and the 5' untranslated region and TATA boxes of Vigun10g019300 and Vigun10g019400, respectively. Three SNPs caused amino acid changes in NAD-ME1 in CSR12906, N299S, S488N and S544N. Protein prediction analysis suggested that S488N of CSR12906 may have a deleterious effect on the function of NAD-ME1. Gene expression analysis demonstrated that IT90K-59-120 and CSR12906 challenged with C. canescens showed different expression in both Vigun10g019300 and Vigun10g019400. Taken together, these results indicated that Vigun10g019300 and Vigun10g019400 are the candidate genes for CLS resistance in the cowpea IT90K-59-120. Two derived cleaved amplified polymorphic sequence markers were developed to detect the resistance alleles at Vigun10g019300 and Vigun10g019400 in IT90K-59-120.

Construction of genetic linkage map and genome dissection of domestication-related traits of moth bean (Vigna aconitifolia), a legume crop of arid areas.

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.

Genome sequence of Jatropha curcas L., a non-edible biodiesel plant, provides a resource to improve seed-related traits.

Jatropha curcas (physic nut), a non-edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development contributes to accumulation of high level of oil in kernels by preventing the breakdown of TAG. A physical cluster of genes for diterpenoid biosynthetic enzymes, including casbene synthases highly responsible for a toxic compound, phorbol ester, in seed cake, was syntenically highly conserved between Jatropha and castor bean. Transcriptomic analysis of female and male flowers revealed the up-regulation of a dozen family of TFs in female flower. Additionally, we constructed a robust species tree enabling estimation of divergence times among nine Jatropha species and five commercial crops in Malpighiales order. Our results will help researchers and breeders increase energy efficiency of this important oil seed crop by improving yield and oil content, and eliminating toxic compound in seed cake for animal feed.

Genetic diversity and structure of the zombi pea (Vigna vexillata (L.) A. Rich) gene pool based on SSR marker analysis.

Zombi pea (Vigna vexillata (L.) A. Rich) is an underutilized legume species and a useful gene source for resistance to biotic and abiotic stresses, although there is little understanding on its genetic diversity and structure. In this study, 422 (408 wild and 14 cultivated) accessions of zombi pea from diverse origins (201 from Africa, 126 from America, 85 from Australia, 5 from Asia and 5 from unknown origin) were analyzed with 20 simple sequence repeat (SSR) markers to determine its genetic diversity and genetic structure. The SSR markers detected 273 alleles in total with a mean of 13.6 alleles per locus. Polymorphism information content values of the markers varied from 0.58 to 0.90 with an average of 0.76. Overall gene diversity was 0.715. Gene diversity and average allelic richness was highest in Africa (0.749 and 8.08, respectively) and lowest in America (0.435 and 4.10, respectively). Nei's genetic distance analysis revealed that the highest distance was between wild Australia and cultivated Africa (0.559), followed by wild West Africa and wild Australia (0.415). STRUCTURE, neighbor-joining (NJ), and principal coordinate analyses consistently showed that these zombi pea accessions were clustered into three major groups, viz. America, Africa and Asia, and Australia. NJ tree also suggested that American and Australian accessions are originated from East African zombi peas, and that the cultivated accessions from Africa and Asia were genetically distinct, while those from America were clustered with some cultivated accessions from Africa. These results suggest that Africa is the center of origin and diversity of zombi pea, and that domestication of this pea took place more than once in different regions.

A gene encoding a polygalacturonase-inhibiting protein (PGIP) is a candidate gene for bruchid (Coleoptera: bruchidae) resistance in mungbean (Vigna radiata).

KEY MESSAGE: The Br locus confers bruchid resistance in mungbean; VrPGIP2 (encoding a polygalacturonase inhibitor) is a strong candidate gene for this resistance. The VrPGIP2 sequence differs between resistant and susceptible lines. Azuki bean weevil (Callosobruchus chinensis) and cowpea weevil (Callosobruchus maculatus) are serious insect pests of mungbean during storage. Bruchid resistance in mungbean is controlled by a single dominant locus, Br. Although the Br locus has been located on a genetic map, molecular basis and function of the gene remain unknown. In this study, high-resolution mapping using a BC11F2 population of 418 plants derived from a cross between 'Kamphaeng Saen 1' (KPS1; susceptible) and 'V2802' (resistant) using simple sequence repeat (SSR) markers delimited the Br locus to a genomic region of 38 Kb of chromosome 5 containing two annotated genes. EST-SSR marker DMB-SSR158 co-segregated perfectly with the Br locus. Bioinformatics analyses revealed that DMB-SSR158 corresponds to a gene encoding a polygalacturonase inhibitor (polygalacturonase-inhibiting protein PGIP) and was designated as VrPGIP2. Comparison of VrPGIP2 coding sequences between four bruchid-resistant (V2802, V1128, V2817 and TC1966) and four bruchid-susceptible (KPS1, Sulu-1, CM and an unknown accession) mungbean lines revealed six single nucleotide polymorphisms (SNPs) between the resistant and susceptible groups. Three of the six SNPs resulted in amino acid changes; namely, alanine (A) to serine (S) at position 320, leucine (L) to proline (P) at position 332, and threonine (T) to P at position 335 of the VrPGIP2 sequence in resistant lines, compared with that in susceptible lines. The A to S change at position 320 may affect the interaction between PGIP and polygalacuronase. These results indicate that VrPGIP2 is very likely the gene at the Br locus responsible for bruchid resistance in mungbean.

A single base substitution in BADH/AMADH is responsible for fragrance in cucumber (Cucumis sativus L.), and development of SNAP markers for the 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.

Genetic diversity of the black gram [Vigna mungo (L.) Hepper] gene pool as revealed by SSR markers.

In this study, 520 cultivated and 14 wild accessions of black gram (Vigna mungo (L.) Hepper) were assessed for diversity using 22 SSR markers. Totally, 199 alleles were detected with a mean of 9.05 alleles per locus. Wild black gram showed higher gene diversity than cultivated black gram. Gene diversity of cultivated accessions among regions was comparable, while allelic richness of South Asia was higher than that of other regions. 78.67% of the wild gene diversity presented in cultivated accessions, indicating that the domestication bottleneck effect in black gram is relatively low. Genetic distance analysis revealed that cultivated black gram was more closely related to wild black gram from South Asia than that from Southeast Asia. STRUCTURE, principal coordinate and neighbor-joining analyses consistently revealed that 534 black gram accessions were grouped into three major subpopulations. The analyses also revealed that cultivated black gram from South Asia was genetically distinct from that from West Asia. Comparison by SSR analysis with other closely related Vigna species, including mungbean, azuki bean, and rice bean, revealed that level of gene diversity of black gram is comparable to that of mungbean and rice bean but lower than that of azuki bean.

QTL mapping for salt tolerance and domestication-related traits in Vigna marina subsp. oblonga, a halophytic species.

QTL mapping in F2 population [V. luteola × V. marina subsp. oblonga] revealed that the salt tolerance in V. marina subsp. oblonga is controlled by a single major QTL. The habitats of beach cowpea (Vigna marina) are sandy beaches in tropical and subtropical regions. As a species that grows closest to the sea, it has potential to be a gene source for breeding salt-tolerant crops. We reported here for the first time, quantitative trait loci (QTLs) mapping for salt tolerance in V. marina. A genetic linkage map was constructed from an F2 population of 120 plants derived from an interspecific cross between V. luteola and V. marina subsp. oblonga. The map comprised 150 SSR markers. The markers were clustered into 11 linkage groups spanning 777.6 cM in length with a mean distance between the adjacent markers of 5.59 cM. The F2:3 population was evaluated for salt tolerance under hydroponic conditions at the seedling and developmental stages. Segregation analysis indicated that salt tolerance in V. marina is controlled by a few genes. Multiple interval mapping consistently identified one major QTL which can explain about 50% of phenotypic variance. The flanking markers may facilitate transfer of the salt tolerance allele from V. marina subsp. oblonga into related Vigna crops. The QTL for domestication-related traits from V. marina are also discussed.

Mapping quantitative trait loci for yield-related traits in soybean (Glycine max L.).

Development of soybean cultivars with high seed yield is a major focus in soybean breeding programs. This study was conducted to identify genetic loci associated with seed yield-related traits in soybean and also to clarify consistency of the detected QTLs with QTLs found by previous researchers. A population of 135 F2:3 lines was developed from a cross between a vegetable soybean line (MJ0004-6) and a landrace cultivar from Myanmar (R18500). They were evaluated in the experimental field of Kasetsart University, Kamphaeng Saen, Nakhon Pathom, Thailand in a randomized complete block design with two replications each in 2011 and 2012 growing seasons. The two parents exhibited contrasting characteristics for most of the traits that were mapped. Analysis of variance showed that the main effects of genotype and environment (year) were significant for all studied traits. Genotype by environment interaction was also highly significant for all the traits. The population was genotyped by 149 polymorphic SSR markers and the genetic map consisted of 129 SSR loci which converged into 38 linkage groups covering 1156 cM of soybean genome. There were 10 QTLs significantly associated with seed yield-related traits across two seasons with single QTLs explaining between 5.0% to 21.9% of the phenotypic variation. Three of these QTLs were detected in both years for days to flowering, days to maturity and 100 seed weight. Most of the detected QTLs in our research were consistent with earlier QTLs reported by previous researchers. However, four novel QTLs including SF1, SF2 and SF3 on linkage groups L and N for seed filling period and PN1 on linkage group D1b for pod number were identified in the present study.

A Cluster of Peronospora parasitica 13-like (NBS-LRR) Genes Is Associated with Powdery Mildew (Erysiphe polygoni) Resistance in Mungbean (Vigna radiata).

Powdery mildew (PM) caused by Erysiphe polygoni is an important foliar disease in mungbean (Vigna radiata). A previous study showed that QTL qPMRUM5-2 is a major locus for PM resistance in mungbean accession RUM5 (highly resistant). Bioinformatics analysis revealed that flanking markers of the qPMRUM5-2 covered a region of 1.93 Mb. In this study, we conducted fine mapping for the qPMRUM5-2 using the F2 population of 1156 plants of the cross between Chai Nat 60 (CN60; highly susceptible) and RUM5. PM resistance evaluation was performed under field conditions using F2:3 lines grown in three different environments. QTL analyses consistently located the qPMRUM5-2 to a 0.09 cm interval on linkage group 6 between InDel markers VrLG6-InDel05 and VrLG6-InDel10, which corresponded to a 135.0 kb region on chromosome 8 containing nine predicted genes of which five were NBS-LRR-type genes Recognition of Peronospora parasitica 13-like protein (RPP13L). Whole-genome re-sequencing of RUM5 and CN60 showed polymorphisms in four RPP13L genes predictively cause substantial amino acid changes, rendering them important candidate genes for PM resistance. The InDel markers VrLG6-InDel05 and VrLG6-InDel10 flanking to the qPMRUM5-2 would be useful for marker-assisted breeding of PM resistance in the mungbean.

A de novo chromosome-scale assembly of the Lablab purpureus genome.

Introduction: Lablab (Lablab purpureus (L.) Sweet), an underutilized tropical legume crop, plays a crucial role in global food and nutritional security. To enhance our understanding of its genetic makeup towards developing elite cultivars, we sequenced and assembled a draft genome of L. purpureus accession PK2022T020 using a single tube long fragment read (stLFR) technique. Results and discussion: The preliminary assembly encompassed 367 Mb with a scaffold N50 of 4.3 Mb. To improve the contiguity of our draft genome, we employed a chromatin contact mapping (Hi-C) approach to obtain a pseudochromosome-level assembly containing 366 Mb with an N50 length of 31.1 Mb. A total of 327.4 Mb had successfully been anchored into 11 pseudomolecules, corresponding to the haploid chromosome number in lablab. Our gene prediction recovered 98.4% of the highly conserved orthologs based on the Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. Comparative analyses utilizing sequence information from single-copy orthologous genes demonstrated that L. purpureus diverged from the last common ancestor of the Phaseolus/Vigna species approximately 27.7 million years ago. A gene family expansion analysis revealed a significant expansion of genes involved in responses to biotic and abiotic stresses. Our high-quality chromosome-scale reference assembly provides an invaluable genomic resource for lablab genetic improvement and future comparative genomics studies among legume species.

Gene discovery and functional marker development for fragrance in sorghum (Sorghum bicolor (L.) Moench).

KEY MESSAGE: 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.

Detection of quantitative trait loci for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean (Vigna radiata (L.) Wilczek) in India and Pakistan.

Yellow mosaic disease (YMD) is one of the major diseases affecting mungbean (Vigna radiata (L.) Wilczek). In this study, we report the mapping of the quantitative trait locus (QTL) for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean. An F8 recombinant inbred line (RIL) mapping population was generated in Thailand from a cross between NM10-12-1 (MYMIV resistance) and KPS2 (MYMIV susceptible). One hundred and twenty-two RILs and their parents were evaluated for MYMIV resistance in infested fields in India and Pakistan. A genetic linkage map was developed for the RIL population using simple sequence repeat (SSR) markers. Composite interval mapping identified five QTLs for MYMIV resistance: three QTLs for India (qYMIV1, qYMIV2 and qYMIV3) and two QTLs for Pakistan (qYMIV4 and qYMIV5). qYMIV1, qYMIV2, qYMIV3, qYMIV4 and qYMIV5 explained 9.33%, 10.61%, 12.55%, 21.93% and 6.24% of variation in disease responses, respectively. qYMIV1 and qYMIV4 appeared to be the same locus and were common to a major QTL for MYMIV resistance in India identified previously using a different resistant mungbean.

A Gene Encoding Xylanase Inhibitor Is a Candidate Gene for Bruchid (Callosobruchus spp.) Resistance in Zombi Pea (Vigna vexillata (L.) A. Rich).

Two bruchid species, Callosobruchus maculatus and Callosobruchus chinensis, are the most significant stored insect pests of tropical legume crops. Previously, we identified a major QTL, qBr6.1, controlling seed resistance to these bruchids in the cultivated zombi pea (Vigna vexillata) accession 'TVNu 240'. In this study, we have narrowed down the qBr6.1 region and identified a candidate gene conferring this resistance. Fine mapping using F2 and F2:3 populations derived from a cross between TVNu 240 and TVNu 1623 (susceptible) revealed the existence of two tightly linked QTLs, designated qBr6.1-A and qBr6.1-B, within the qBr6.1. The QTLs qBr6.1-A and qBr6.1-B explained 37.46% and 10.63% of bruchid resistance variation, respectively. qBr6.1-A was mapped to a 28.24 kb region containing four genes, from which the gene VvTaXI encoding a xylanase inhibitor was selected as a candidate gene responsible for the resistance associated with the qBr6.1-A. Sequencing and sequence alignment of VvTaXI from TVNu 240 and TVNu 1623 revealed a 1-base-pair insertion/deletion and five single-nucleotide polymorphisms (SNPs) in the 5' UTR and 11 SNPs in the exon. Alignment of the VvTAXI protein sequences showed five amino acid changes between the TVNu 240 and TVNu 1623 sequences. Altogether, these results demonstrated that the VvTaXI encoding xylanase inhibitor is the candidate gene conferring bruchid resistance in the zombi pea accession TVNu 240. The gene VvTaXI will be useful for the molecular breeding of bruchid resistance in the zombi pea.

Mapping of quantitative trait locus reveals PsXI gene encoding xylanase inhibitor as the candidate gene for bruchid (Callosobruchus spp.) resistance in pea (Pisum sativum L.).

Pea (Pisum sativum L.) is an important legume crop for both food and feed. Bruchids (Callosobruchus spp.) are destructive insect pests of pea in the field and during storage. In this study, we identified a major quantitative trait locus (QTL) controlling seed resistance to C. chinensis (L.) and C. maculatus (Fab.) in field pea using F2 populations derived from a cross between PWY19 (resistant) and PHM22 (susceptible). QTL analysis in the two F2 populations grown in different environments consistently identified a single major QTL, qPsBr2.1, controlling the resistance to both bruchid species. qPsBr2.1 was mapped onto linkage group 2 between DNA markers 18339 and PSSR202109 and explained 50.91% to 70.94% of the variation in resistance, depending on the environment and bruchid species. Fine mapping narrowed down qPsBr2.1 to a genomic region of 1.07 Mb on chromosome 2 (chr2LG1). Seven annotated genes were found in this region, including Psat2g026280 (designated as PsXI), which encodes a xylanase inhibitor and was considered as a candidate gene for bruchid resistance. PCR amplification and sequence analysis of PsXI suggested the presence of an insertion of unknown length in an intron of PWY19, which causes variation in the open reading frame (ORF) of PsXI. Moreover, the subcellular localization of PsXI differed between PWY19 and PHM22. These results together suggested that PsXI encoding xylanase inhibitor is responsible for the bruchid resistance of the field pea PWY19.

Fine mapping of QTL conferring resistance to calcareous soil in mungbean reveals VrYSL3 as candidate gene for the resistance.

Iron is a crucial nutrient for biological functions in plants. High-pH and calcareous soil is a major stress causing iron deficiency chlorosis (IDC) symptoms and yield losses in crops. Use of calcareous soil-tolerance genetic resources is the most effective preventative method to combat the effects of high-pH and calcareous soils. A previous study using a mungbean recombinant inbred line (RIL) population of the cross Kamphaeg Saen 2 (KPS2; IDC susceptible) × NM-10-12 identified a major quantitative trait locus (QTL), qIDC3.1, which controls resistance and explains more than 40% of IDC variation. In this study, we fine-mapped qIDC3.1 and identified an underlying candidate gene. A genome wide association analysis (GWAS) using 162 mungbean accessions identified single nucleotide polymorphisms (SNPs) on chromosome 6; several SNPs were associated with soil plant analysis development (SPAD) values and IDC visual scores of mungbeans planted on calcareous soil, respectively. These SNPs corresponded to qIDC3.1. Using the same RIL population as in the previous study and an advanced backcross population developed from KPS2 and IDC-resistant inbred line RIL82, qIDC3.1 was further confirmed and fine-mapped to an interval of 217 kilobases harboring five predicted genes, including LOC106764181 (VrYSL3), which encodes a yellow stripe1-like-3 (YSL3) protein, YSL3 is involved in iron deficiency resistance. Gene expression analysis revealed that VrYSL3 was highly expressed in mungbean roots. In calcareous soil, expression of VrYSL3 was significantly up-regulated, and it was more obviously upregulated in the roots of RIL82, than in those of KPS2. Sequence comparison of VrYSL3 between the RIL82 and KPS2 revealed four SNPs that result in amino acid changes in the VrYSL3 protein and a 20-bp insertion/deletion in the promoter where a cis-regulatory element resides. Transgenic Arabidopsis thaliana plants overexpressing VrYSL3 showed enhanced iron and zinc contents in the leaves. Taken together, these results indicate that VrYSL3 is a strong candidate gene responsible for calcareous soil resistance in mungbean.