​Association of ISSR and ISSR-RGA Markers with Powdery Mildew Resistance Gene in Mungbean

Mungbean [Vigna radiata (L.) Wilczek] serves as an important food legume crop due to its highly nutritious benefits for human health and it is grown over 7 million hectares, mostly in temperate, tropical and subtropical zones of East, South and Southeast Asia (Yu et al., 2011; Nair et al., 2019). In general, its yield potential ranges from 2.5-3.0 t/ha, whereas the total production is low, being about 0.5 t/ha (Nair et al., 2019). Powdery mildew (PM) constitutes one of the major constraints to global mungbean production. Sphaerotheca phaseoli is severe biotrophic parasite causing PM disease on the lower and upper leaf surfaces of mungbean, particularly widespread in the cool dry season. Although there has been an attempt to control the disease by fungicides, it is not only responsible for increased costs, but it is also severely harmful to human health and the environment. Alternatively, there has long been interest in developing PM resistant varieties for sustainable management of the disease.
       
Understandably, plant resistance genes (R) are either qualitative or quantitative in nature. Qualitative resistance is monogenic by a single gene or a few genes and typically is race specific, while quantitative resistance is polygenic and race non-specific (Barakat et al., 2008). Previously, our observations revealed that resistance to PM in three Indian mungbean accessions from the World Vegetable Center, Taiwan, namely V4718, V4758 and V4785 showing highly and stable resistance to PM resulted from a single dominant gene with independent segregation and these monogenic resistance genes could be pyramided to develop a resistant variety with broad spectrum resistance (Khajudparn et al., 2010).
       
Molecular markers tightly linked to any gene of interest which are regarded as steady landmarks have long been reported to be an effective tool for plant breeders to indirectly select promising plants, particularly those obtained from pyramiding multiple resistance genes (Kour et al., 2011). In particular, inter-simple sequence repeat (ISSR) markers which implicate the use of a single SSR-containing primer for direct amplification of sequences at multiple loci are highly efficient, easy, economical and rapid. In many reports, ISSR markers have been more accurate and consistently polymorphic than randomly amplified polymorphic DNA (RAPD) markers as well as being effective for tagging disease resistance genes (Ali et al., 2011; Bainade et al., 2014; Tantasawat et al., 2020). In addition, resistance gene analog (RGA) markers, which were first reported by Chen et al., (1998) are resistance-gene based markers amplified from the highly conserved structures of genes involved in plant defense mechanism. These structures involve recognition of unique avirulence (Avr) factors and then induce signaling and programmed cell death, respectively. It is interesting to note that a large number of R genes, which share several conserved domains i.e., leucine-rich repeat (LRR), nucleotide-binding sites (NBS) and protein kinase domain, implicate resistance against a large spectrum of pathogens. These conserved domains have been found in genes isolated from genomes of several plant species i.e., tomato, eggplant and legume crops (Wei et al., 2016; Dev et al., 2018; Tirnaz et al., 2020). Recently, we developed a new type of marker, ISSR-anchored resistance gene analog (ISSR-RGA), using ISSR primers combined with gene-targeted RGA primers to detect the SSRs dispersed at multiple loci in proximity to RGAs throughout the mungbean genome and found that the ISSR-RGA markers are more effective than ISSRs for successfully identifying the gene conferring PM resistance in the ‘CN72 × V4718’ cross (Poolsawat et al., 2017). Therefore, we extend the uses of ISSR-RGA markers along with ISSR markers for identifying markers associated with another PM resistance gene in the ‘CN72 × V4785’ cross.

Plant materials
 
In this study, population of 126 F_2:9 recombinant inbred lines (RILs) developed from CN72 and V4785 was used. The donor parent V4785 is highly resistant to PM at all growth stages, while the recipient parent CN72 is popularly grown by Thai farmers and is highly susceptible to PM (Khajudparn et al., 2010).
 
Phenotypic evaluation
 
The F_2:9 RIL population and their parents were planted in a randomized complete block design (RCBD) with three replications during the winter season at Suranaree University of Technology farm, Nakhon Ratchasima, Northeast Thailand in 2015 and 2018. For each plot, seeds were sown in two-meter-long rows with a spacing of 0.5 and 0.2 m between rows and between plants within rows, respectively. Susceptible spreader rows were also planted as the important sources of PM inoculum. The plant reaction to PM was rated in rows at 65 days after planting using a relative scale of 1-9 according to Khajudparn et al., (2010). The average scores were divided into two categories (resistant and susceptible individuals with scores rating 1-4.9 and 5-9, respectively). The distribution of PM resistance was assessed with the chi-square test (c²). Scores of PM response in both years were transformed with the (X+1)^1/2 formula for analyzing the broad sense heritability (h²b) as described by Khajudparn (2009). The SPSS version 14.0 (Levesque, 2006) was used to determine the relationship of PM resistance in both years through correlation analysis.
 
ISSR and ISSR-RGA analysis
 
Young leaf samples of three-week-old plants were collected and DNA isolation was conducted using a modified CTAB extraction protocol (Lodhi et al., 1994). For polymerase chain reaction (PCR) amplification, the concentration and purity of the extracted DNA were determined spectro- -photometrically at A₂₆₀ and A₂₈₀ by Nanodrop ND-1000 (NanoDrop Technologies, Inc., Wilmington, DE, USA). A total of 63 ISSR primers developed from the University of British Columbia and 241 ISSR-RGA primer combinations that were derived from 63 ISSR primers and 4 RGA primers complementary to the conserved structures of R genes including NBS (GLPLAL 1 and P-Loop; Mahanil et al., 2007) and kinase domains (Pto kin 1; Chen et al., 1998 and RLK for; Feuillet et al., 1997) were used to screen two parents (R and S) and two different bulks, each containing 10 RIL informative individuals showing either the highest resistance (resistant bulk; RB) or susceptibility (susceptible bulk; SB), according to bulk segregant analysis (BSA). The sequences of these primers are shown in Tables 1 and 2. PCR and DNA banding visualization for both marker systems were totally performed according to Poolsawat et al., (2017). Comparison of DNA banding patterns between R and S as well as RB and SB was carried out to identify putative ISSR and ISSR-RGA loci for the PM resistance.
 

 

 
Marker-trait analysis
 
For marker genotyping, the clearly resolved DNA profiles were scored manually by assigning ‘0’ and ‘1’ for their absence and presence of each polymorphic band, respectively. The association between the putative marker and PM resistance was analyzed through a simple linear regression using the SPSS version 14.0 (Levesque, 2006) and the logarithm of odd (LOD) analysis. Magnitude of the marker associated phenotypic effects was explained by the coefficient of determination (R²), of which polymorphism of the marker represented the fraction of variance.

Inheritance information of powdery mildew resistance
 
The donor parent V4785 was immune to natural PM infection with the disease scores of 1.33 and 1.00 in 2015 and 2018, respectively, while the recipient parent CN72 was highly susceptible to the disease with the disease scores of 6.33 and 6.00 in 2015 and 2018, respectively (Fig 1a and 1b). Among the F_2:9 RILs consisting of 126 individuals, 61 and 65 individuals exhibited resistant and susceptible response, respectively in 2015, while 53 and 73 individuals exhibited resistant and susceptible response, respectively in 2018. The disease scores of both years were significantly correlated (r = 0.61, P<0.001). The c² test revealed that the population phenotypically segregated into a ratio of 1:1 (resistant/susceptible) in both years (c² = 0.13 and 3.17, respectively, P_0.05 = 3.84). The estimates of broad sense heritability for PM resistance in 2015 and 2018 were 79.57 and 89.36%, respectively. The results demonstrated that the PM resistance in V4785 was due to a single major gene. This inheritance pattern was also previously observed by Khajudparn et al., (2010) who reported that three mungbean resistant lines, namely V4718, V4758 and V4785 inherited PM resistance genes as single genes with nonallelic interaction. In addition, many studies also reported that dominant gene actions implicated in inheritance of PM resistance in different sources of mungbean (Reddy et al., 1994; Humphry et al., 2003). On the other hand, Kasettranan et al., (2010) found that PM resistance was quantitatively inherited in F₇ population of the ‘KPS1 × VC6468-11-1A’ cross. Therefore, it is important to note that PM resistance in various resistant sources may be inherited differently.
 

 
ISSR and ISSR-RGA analysis
 
To identify marker loci for the PM resistance gene, a total of 63 ISSR primers and 241 ISSR-RGA primer combinations were screened in BSA. It was found that the 63 ISSR primers amplified a total of 1,260 DNA bands with an average of 20 bands/ primer. Of these, 75 were found to be polymorphic between CN72 and V4785, giving a polymorphism percentage of 5.95. Only 40 of 63 primers gave polymorphic bands between parents with percentages of polymorphism ranging from 2.94 to 28.57% and two of these polymorphic bands from ISSR 808 and 827 were identified as the putatively linked marker loci for PM resistance (Table 1). The 63 ISSR primers were used again to combine with 4 RGA primers for identification of the PM resistance gene. The results revealed that 241 ISSR-RGA primer combinations could produce clear bands. However, only 28 out of 241 primer combinations, which amplified a total of 810 DNA bands with an average of 28.93 bands/ primer pair produced polymorphic DNA bands. In total, 96 polymorphic DNA bands were observed with an average of 3.43 bands/ primer pair and 11.85 polymorphism percentage (Table 2). Among these polymorphic loci, the ISSR and ISSR-RGA marker-based BSA revealed 2 and 37 markers, respectively, whose specific bands were exclusively present in resistant parent and resistant bulk/or susceptible parent and susceptible bulk, indicating their possible association with the PM resistance gene. BSA was proved to be a very useful tool for rapid identification of marker loci linked to any disease resistance genes (Uma et al., 2016). To identify more closely linked markers, screening of more primers is required to obtain sufficient polymorphism in the target region through BSA. In this study, screening of 63 ISSR primers and 241 ISSR-RGA primer combinations was rapidly conducted to identify more closely linked markers using BSA and we found that 2 ISSR and 37 ISSR-RGA markers were putatively associated with the PM resistance. Using this analysis, Bainade et al., (2014) also successfully identified a marker from ISSR 834 that was associated with the PM resistance gene in the ‘Kopargaon × BPMR-48’ cross of mungbean.
       
Considering 2 ISSR and 37 ISSR-RGA markers as putatively associated with PM resistance from BSA, all of these markers were subsequently tested using simple linear regression and LOD analysis. One out of two ISSR markers (I27565; P<0.01) and three out of 37 ISSR-RGA markers (I10P321, I27R211 and I27R565; P<0.01) were significantly associated with the PM resistance gene that a coefficient of determination (R²) of 0.560 for the I27565 marker and 0.479, 0.723 and 0.560 for the I10P321, I27R211 and I27R565 markers, respectively was found (Table 3). However, the I27565 and I27R565 markers localized on the same locus and all four markers (I27565, I10P321, I27R211 and I27R565) resided on the same side relative to the PM resistance gene. Three of these markers had LOD scores  of above 3, passing threshold after permutation. The I27R211 and I27R565 markers in Fig 2, which were amplified by the same primer pair (ISSR827 combined with RLK for) and were scored simultaneously were closest to the PM resistance gene (5 and 10 cM, respectively). The position of both markers (I27R211 and I27R565) in the current ‘CN72 × V4785’ cross studied was different from those of I42PL229 and I85420 markers flanking the PM resistance gene in the ‘CN72 × V4718’ cross of mungbean (Poolsawat et al., 2017), confirming the nonallelic nature of the PM resistance genes in V4718 and V4785. These results suggest the possibility of developing a variety with broad spectrum resistance to PM through marker-assisted gene pyramiding in mungbean breeding programs.
 

 

The donor resistant line V4785 has conferred a qualitative resistance for PM through a single dominant gene. The I27R211 and I27R565 ISSR-RGA markers closest to this resistance gene will be applicable for MAS of a PM resistant mungbean that allow pyramiding of multiple PM resistance genes to provide more durable and sustainable resistance in the future. Moreover, we also verify the usefulness of ISSR-RGA marker system for tagging a disease resistance gene.

This work was supported by Suranaree University of Technology (SUT) and by Thailand Science Research and Innovation (TSRI). The World Vegetable Center, Taiwan and Chai Nat Field Crops Research Center, Thailand are gratefully acknowledged for providing the seeds of V4785 and CN72, respectively. We are also thankful to Mr. Peter C. Bint for proofreading the manuscript.