Mungbean [Vigna radiata (L.) Wilczek] is an important legume crop of Asia. It is cultivated on ~6 million hectares, mainly in Asia (Somta et al., 2007). The crop is popularly grown for seeds after rice or wheat because of its early maturity (60–75 day), drought tolerance and symbiotic relationship with rhizobia, which fix atmospheric nitrogen to the soil. Seeds of mungbean contain high levels of proteins (20–25%) and carbohydrate (60–75%), and thus, they are an important human dietary staple (Somta et al., 2007).

Bruchids or seed weevils (Coleoptera: Bruchidae) are serious pests that damage legume seeds after harvest. Bruchids infest seeds in the field and after harvest, and the latter can results in total loss of a seed lot within 3–4 months (Srinives et al., 2007). The bruchid-damaged seeds are inedible and cannot be used for agricultural and commercial purposes. Azuki bean weevil (Callosobruchus chinensis L.) and cowpea weevil (Callosobruchus muculatus F.) are the major bruchid species that feed on seeds of mungbean and other food legumes, including cowpea (Vigna unguiculata L.), black gram [Vigna mungo (L.) Hepper], Bambara groundnut [Vigna subterranea (L.) Verdc.], azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi], soybean [Glycine max (L.) Merr.], chickpea (Cicer arietinum L.) and pigeon pea [Cajanus cajan (L.) Millsp.]. Although C. chinensis and C. maculatus originated in Asia and Africa, respectively, they are currently present in almost every continent owing to international seed/grain trading. Generally, bruchids are controlled by chemical fumigation and dusting. However, these chemicals are not only harmful to humans and the environment, but also increase production costs. Additionally, chemical control is not practical for small-landholder farmers. Thus, a major goal in mungbean breeding programs is to develop bruchid-resistant cultivar(s) (Somta et al., 2007; Srinives et al., 2007).

Several bruchid-resistant mungbean germplasms have been identified. Fujii and Miyazaki (1987) reported that wild mungbean (V. radiata var. sublobata) accession TC1966 is completely resistant to C. chinensis and C. maculatus. Talekar and Lin (1992) found that cultivated mungbean accessions V2709 and V2802 are highly resistant to C. chinensis. Somta et al. (2008) demonstrated that V2709 and V2802 are also highly resistant to C. maculatus, while the cultivated mungbean accessions V1128 and V2817 are completely resistant to both bruchids.

Kitamura et al. (1988) showed that resistance to C. chinensis in “TC1966” is controlled by a single dominant gene, Br. Somta et al. (2007) demonstrated that resistance to C. chinensis and C. maculatus in “V2709” and “V2802” is controlled by a single dominant gene with some modifiers. Kaga and Ishimoto (1998) fine mapped the Br gene in “TC1966” to a region of 0.7 cM between restriction fragment length polymorphism (RFLP) markers Bng110 and Bng143. The Br gene is only 0.2 cM away from the Bng143. Chotechung et al. (2011) found that expressed sequence tag-simple sequence repeat (EST-SSR) marker DMB-SSR158 (DMB158 in the original report) co-segregated perfectly with the Br gene in “V2802”. Later, Chotechung et al. (2016) narrowed the genome region of the Br gene to a 38-kb segment on chromosome 5 and found that a gene, VrPGIP2, encoding polylacturonase-inhibiting protein (PGIP; also known as polylacturonase inhibitor) was probably responsible for the bruchid resistance. They also showed that “V2802”, “V1128”, “V2817”, and “TC1966” had the same VrPGIP2 allele.

For “V2709”, the mode of bruchid-resistance inheritance is the same as for “V2808” and “TC1966” (Somta et al., 2007). However, the EST-SSR marker DMB-SSR158, which is located on the VrPGIP2 sequence, did not reveal a polymorphism between “V2709” and bruchid-susceptible mungbean “Kamphaeng Saen 1” (KPS1; Chotechung et al., 2011), which was the same susceptible parent used by Chotechung et al. (2016). This suggested that the gene or allele for bruchid resistance in “V2709” is different from that of “V2802”.

In this study, we report the identification of new alleles for bruchid resistance in mungbean accession V2709. The objectives of this study were to (i) fine map the Br locus in “V2709”, (ii) identify candidate gene(s) for the Br locus in “V2709” and (iii) identify mutation(s) responsible for the resistance.

Although the wild mungbean accession TC1966, which carries the Br locus that confers resistance to C. chinensis and C. maculatus, has been reported since 1987 (Fujii and Miyazaki, 1987), only a few cultivars possessing the Br locus have been released to farmers (Lee et al., 2000; Yao et al., 2015). This is mainly due to skepticism about the safety of the Br locus. The chemical responsible for bruchid resistance at the Br locus is not known, and toxicity studies of the bruchid-resistant mungbeans carrying this locus are very limited (Miura et al., 1996; Yao et al., 2015). Kitamura et al. (1990) reported that the chemical involved in bruchid resistance was heat-stable and possibly a polysaccharide. Cyclo-peptide alkaloids, called Vignatic acid A and Vignatic acid B, isolated from a bruchid-resistant breeding line were detrimental to C. chenensis (Sugawara et al., 1996). A cysteine-rich protein designated VrCRP/VrD1 (defensin) identified from a bruchid-resistant breeding line inhibited the development of C. chenensis larva (Chen et al., 2002). However, gene mapping (Kaga and Ishimoto, 1998) and linkage analyses (Isemura et al., 2012) revealed that the loci controlling these chemicals are not in the Br locus.

Chotechung et al. (2016) demonstrated that VrPGIP2, which encodes a PGIP, is the candidate gene for the Br locus in mungbean “V2802”, although a role for VrPGIP1, which also encodes a PGIP and is located next to VrPGIP2, could not be eliminated. VrPGIP2 in mungbean may confer bruchid resistance by inhibiting the digestive polygalacturonases (PGs) of bruchids that degrade pectin in the seeds, producing an energy source for the bruchids and helping other digestive enzymes to gain access to their substrates in bruchid food (Chotechung et al., 2016). In this study, we located a single major QTL for bruchid resistance with a high genetic effect (phenotypic variance explained (PVE) > 95%) on chromosome 5 using mungbean accession “V2709”. The genomic confidence region of the QTL (VrID5 to VrBr-SSR037) covered ~237.35 Kb (Vr05: 5,410,272–5,647,621) with eight protein-encoding genes, including VrPGIP1 and VrPGIP2 (Figure 1 and Supplementary Table S2).

We sequenced VrPGIP1 and VrPGIP2 in mungbean “V2709” and found new alleles, VrPGIP1-1 and VrPGIP2-2, respectively (Figures 3A, 4). The VrPGIP2 encoded by VrPGIP2-2 possessed an amino acid change at the position 185, as compared with the wild type protein (Figure 3B). Comparing the sequence of VrPGIP2 with PvPGIP2, which is the only PGIP that has a known crystal structure, we found that residue 185 of the VrPGIP2 primary sequence (Figure 6B) corresponds to residue 164 of the crystal structure of PvPGIP2 (Di Matteo et al., 2003). Residue 164 is a site that contributes to the formation of the secondary structure of the PvPGIP2 protein (Di Matteo et al., 2003), and it is also a putative positive selection site of PvPGIP2 that plays an important role in PG recognition (Casasoli et al., 2009). The VrPGIP1 encoded by VrPGIP1-1 showed many amino acid changes when compared with the wild type, and one change that occurred at position 185, which is the same position of the single amino acid change in VrPGIP2, resulted in the same amino acid at this position in VrPGIP1 and VrPGIP2 in “V2709” (Figures 5, 6C). PGs are important digestive enzymes found in the midgut of Callosobruchus maculatus (Pedra et al., 2003; Pauchet et al., 2010; Nogueira et al., 2012; Santana, 2013) and Phaedon cochleariae (mustard leaf beetle) (Kirsch et al., 2012), a coleopteran insect similar to the Callosobruchus species. A semi-purified PGIP from orange flavedo inhibited an endoPG purified from the larvae of sugarcane rootstalk borer weevil (Diaprepes abbreviates) (Doostdar et al., 1997), while PvPGIP3 and PvPGIP4 from the common bean strongly inhibited the PG activities of the mirid bugs Lygus rugulipennis and Adelphocoris lineolatus (D'Ovidio et al., 2004; Frati et al., 2006). Insect pectinase complexes, including PGs, are mainly found in Coleopteran and Hemipteran insects and are involved in plant penetration, the softening of plant material, and the digestion of young plant tissues and grains (Calderón-Cortés et al., 2012). Kirsch et al. (2016) showed that the rice weevil Sitophilus oryzae, a coleopteran insect that infests both cereal and legume seeds, uses PGs and pectin methylesterases synergistically to degrade pectin. If this was also the case in C. chinensis and C. maculatus, VrPGIP2-2 and also VrPGIP2-1 could confer resistance to these bruchids by inhibiting the ability of PG to degrade pectin, which would result in the retardation and arrest of insect growth and development.

Hong et al. (2015) demonstrated that two closely linked QTLs (~7-cM apart) with similar effects (PVE for each QTL was ~45%) controlled C. chinesis resistance in “V2709” although a genetic study revealed the monogenic inheritance of the trait. This contradicts our finding that only one single major QTL with a PVE greater than 98% on mungbean chromosome 5 controlled bruchid resistance in “V2709”. It is difficult to compare the results between the two studies because only one marker (GBssr-MB87) is common and it is far from the QTLs in both studies. Although the RP marker that is closely linked to a QTL reported by Hong et al. (2015) showed a polymorphism between the parents of our mapping population, it produced weak DNA bands that were difficult to unambiguously score. Nonetheless, the probable location of RP on chromosome 5 is between 5,210,000 and 5,520,000 (Chotechung et al., 2016).

In addition to VrPGIP genes that show an association with bruchid resistance (this study and Chotechung et al., 2011), g39185 encoding a BURP domain-containing protein has been identified as a candidate gene for bruchid resistance in mungbean. g39185 was identified by comparing transcript and protein profiles between bruchid-susceptible and bruchid-resistant mungbean, which derived its resistance from “TC1966” (Lin et al., 2016). g39185 is at position 5,236,101 of mungbean chromosome 5 (Lin et al., 2016) which is ~326.7 and ~354.7 Kbp from VrPGIP1 and VrPGIP2, respectively, and is very near the possible location of the RP marker reported by Hong et al. (2015). BURP domain-containing proteins have only been found in plants. Although many BURP proteins have been isolated from plants, their expression patterns are diverse and some functions are still unknown. Generally, BURP domain-containing proteins play important roles in maintaining normal plant metabolism or development (Shao et al., 2011; Tang et al., 2014; Li et al., 2016). Based on the information reported by Lin et al. (2016), g39185 corresponds to Vradi05g03810 on the reference mungbean chromosome 5, and Vradi05g03810 is annotated as LOC106759697 in the GenBank database. LOC106759697 encodes a PG (https://www.ncbi.nlm.nih.gov/gene/?term$=$LOC106759697). Three forms of PGs were predicted from LOC106759697. A BLASTN algorithm-based search revealed that the gene was also most similar to a gene from azuki bean (e-value = 0.0, identity = 95%) that encodes a RESPONSIVE TO DEHYDRATION22 (RD22)-like protein of the BURP family. The RD22 protein is responsive to abiotic stresses, such as drought and salinity (Abe et al., 2003; Hanana et al., 2008). Based on the known and/or possible physiological function of PGIP and our results from gene sequencing and mapping in this study and in Chotechung et al. (2016), VrPGIP2 and/or VrPGIP1 is most likely the Br locus. Nonetheless, our results in this study indicated that the tightly linked genes VrPGIP1 and VrPGIP2 are likely the genes at the Br locus that confer bruchid resistance in the mungbean “V2709”. In addition, the different genetic bases of the bruchid resistance under the Br loci in “V2709” and “V2802” provides plant breeders the opportunity to develop new mungbean cultivar(s) with durable resistance to bruchids by pyramiding or rotating the VrPGIP1 and VrPGIP2 resistance genes.

AnK and AlK carried out DNA marker analysis and data analysis. PrS, AnK, TY, and PeS prepared plant materials and conducted insect resistance evaluation. JC, XY, and XC performed DNA sequencing, and analyzed sequencing results. PrS initiated and coordinated the study, and analyzed the results. AnK, JC, PrS, XC, and PeS wrote the manuscript. All authors approved the manuscript.