Quantitative Trait Loci (QTL) Detection and Validation of Linked Markers Associated with Yellow Mosaic Virus Resistance in Urdbean [Vigna mungo (L.) Hepper] in Andhra Pradesh

Blackgram [Vigna mungo (L.) Hepper] or urdbean is an important short duration self pollinated pulse crop belongs to family Leguminaceae cultivated in almost all parts of India. In term of area and production, blackgram is the fourth most important cultivated grain legume crop in India after chickpea, pigeonpea and mungbean. In blackgram, yellow mosaic disease (YMD) cause severe yield loss, it is caused by yellow mosaic virus (YMV) belongs to family Geminiviridae, genus begomovirus and YMV is transmitted by whitefly (Bemisia tabaci) from infected to healthy plants. In India, the YMD on blackgram was first reported by Williams et al., (1968). Association of Mungbean yellow mosaic India virus (MYMIV) with YMD of blackgram in Andhra Pradesh was reported by Reddy et al., (2015) for the first time from South India. Among the begomoviruses, MYMV and MYMIV cause severe yield loss up to 85 per cent (AVRDC, 1998), causes the yellow mosaic disease (YMD) on all pulse crops such as greengram, blackgram, soybean, mothbean, cowpea, pigeon pea, common bean, horsegram and dolichos (Mishra et al., 2020).

Management of YMD is often linked with control of the Bemisia tabaci population by spraying insecticides, which is sometimes ineffective because of high population pressure. The vector management using chemicals is expensive and numbers of sprayings are required to control whitefly. Repeated sprayings cause health hazards so, cultivation of virus resistant varieties are the most economical, efficient and environment friendly. Molecular marker technology can enhance the efficiency of breeding through marker-assisted selection (MAS), in which phenotypic selection is carried out together with molecular markers that are associated with the target trait. MAS can accelerate the development of new cultivars by reducing the number of generations and increasing the accuracy in phenotypic evaluations (Collard and Mackill, 2008).

Identification of molecular markers tightly linked with the trait of interest is one of the prerequisites for transfer of QTL(s)/gene(s) for YMV resistance employing marker assisted breeding. As of now, a few QTLs have been identified for YMV resistance in urdbean, their direct utility in the MAS programs is limited by their co-inheritance ability with the resistant phenotype in the segregating mapping populations.

The identified molecular markers that are linked with the trait of interest in one population should be validated by testing for the presence of the marker in a range of genotypes. Hence, before using reported markers directly in MAS programs, it is better to validate them in known resistant and susceptible genotypes. Validation of molecular marker linked to YMV disease resistance genes has been reported in urdbean (Prasanthi et al., 2013; Sowmini and Jayamani, 2014; Gupta et al., 2015). Molecular markers linked to YMV resistance also have been reported in mungbean (Kabi et al., 2018).  The objectives of this study were construction of genetic linkage map using SSR markers, detection of QTLs associated to YMV resistance and validation of QTL linked markers that are associate with YMV resistance in blackgram.

Parental polymorphic study and development of F1 individuals
 
The F₁ individuals were developed from the cross between the susceptible genotype (PGB32) and the resistant genotype (PU31) during kharif, 2016 under net house at RARS, Tirupati. Parental polymorphic study was done by employing a total of 147 markers developed from azukibean and greengram genome sequences (Chaitieng et al., 2006; Gupta et al., 2008).
 
Phenotyping and genotyping of F2mapping population
 
The F₁s seeds were selfed to produce F₂ population during rabi, 2017 after confirming their heterozygosity using CEDG180 marker. In total, 180 F₂ individuals of PU31×PBG32 cross along with the parents were evaluated for YMV resistance at RARS, Tirupati during summer, 2018 under natural field conditions. The data for the presence or absence of the YMV disease was recorded from initial flowering to harvesting with 10 days intervals.

The total genomic DNA was isolated from tender leaves of parents, F₁ and F₂ mapping population at 30-40 DAS (days after sowing) using modified CTAB method (Murray and Thomson, 1980). DNA was subjected to PCR by using microsatellite markers and the PCR product was loaded on to 3% agarose gel by mixing with 3 μl of 6 x loading dye, banding pattern was analyzed using gel documentation system (Biorad Gel Doc x R+ Imaging Systems). The genotyping of the F₂ mapping population was carried out using the polymorphic markers identified between the parents. The allele sizes of PCR products were resolved in agarose gel and assigned an allele code of ‘A’ for parent 1 allele (PU31), ‘B’ for parent 2 allele (PBG32) and ‘H for heterozygote to score the entire F₂ mapping population.
 
QTL analysis
 
The genotypic data of cross PU31 x PBG32 generated by polymorphic markers were used for construction of genetic linkage map using ICI mapping software ver. 4 (http://www.isbreeding.net).The generated linkage map and phenotypic data were subjected to Composite Interval Mapping (CIM) for detection of QTLs employing ICImapping software ver. 4 (http://www.isbreeding.net).
 
Validation of QTL linked markers in known blackgram resistant and susceptible genotypes
 
The identified markers flanked to qYMV were validated in 25 diverse resistant and susceptible blackgram genotypes made available from Pulses Breeding Scheme, RARS, Tirupati. These were grown at RARS Farm, Tirupati during summer, 2020. The phenotypic data of blackgram genotypes i.e., susceptible and resistant reaction to YMV disease was recorded. The genomic DNA was isolated from each genotype using CTAB method. For validation of linked markers, the total DNA was amplified in PCR with linked markers that are identified in this study.

Parental polymorphism study using SSR markers
 
A total of nine F1 seeds of crosses PU31 x PBG32 were obtained and sown in the field of RARS, ANGRAU, Tirupati for development of F2 population. Of 147 SSR markers used, 24 showed polymorphism (16%) between resistant and susceptible parents viz., PU31 and PBG32. Gupta et al., (2013) recorded 8.6% of polymorphism between DPU88-31 (YMV resistant) and AKU9904 (YMV susceptible) and Rambabu et al., (2018) reported 28% polymorphism between the resistant (T₉) and susceptible (LBG759) parents of blackgram. Based on these reports it can be implied that low polymorphism is common due to the existence of narrow genetic base in blackgram (Kalaria et al., 2014). However, in our study the SSR markers showed 16% polymorphism that implies availability of higher variation among the parents when compared to the earlier studies.
 
Phenotyping and genotyping of F1 and F2 mapping population
 
Screening of F₁ plants (PU31 x PBG32) showed susceptibility to YMV indicating recessive nature of YMV tolerance. In contrast, Thamodhran et al., (2016) studied inheritance pattern of YMV resistance, wherein three susceptible blackgram genotypes i.e., MDU 1, CO6 and LPG-752 were crossed with four resistant genotypes i.e., VBN (Bg) 6, PU 31, Mash-114 and Uttara. The F1s of all crosses showed resistance to YMV, which means resistance is dominant over the susceptible.

The phenotypic data of F2 population, for the YMV disease reaction i.e., resistance or susceptible nature of each plant was recorded from 30 DAS to harvesting.Of 186 F2 plants of PU31 x PBG32 cross, 152 plants showed disease symptoms, absent in 31 plants and 3 plants were died. The segregation ratio in F₂ population of PU31 x PBG32 cross was 5 susceptible: 1 resistant indicated that MYMV resistance was governed by several genes. Genotypic analysis of F₂ population derived from the cross PU31×PBG32 was performed employing 24 polymorphic SSR markers. The total DNA was amplified in PCR using polymorphic primers, which produced 3 types of alleles among F₂ individuals.

Lekhi et al. (2018) investigated the mode of inheritance of MYMV resistance in the cross SML668 x Mash114 of mungbean reported segregation ratio in F₂ population was 3 resistant: 1 susceptible and Muraleedhar et al., (2015) reported the segregating ratio 9 S: 3 MS: 3 MR: 1 R in F2 population of mungbean.
 
Mapping of YMV resistance genomic regions/QTLs
 
The genotypic data generated employing 18 polymorphic markers was used for construction of genetic linkage map using ICImapping software ver. 4 (http://www.isbreeding.net) at LOD threshold score of 3.0. Of these 17 markers clustered into a single linkage group while one marker namely CEDG225 was unlinked. The total map length observed was 335.47 cM. The highest interval i.e., 41.13 cM observed between the markers CEDG006 and CEDG180 while the lowest interval of 13.62 cM observed between CEDG172 and BM170 markers. Gupta et al., (2008) worked on construction of blackgram genetic linkage map using a total of 428 markers including 254 AFLP, 47 SSR, 86 RAPD and 41 ISSRs at a LOD score of 5.0 and distributed into 11 linkage groups with 865.1cM of total length. In the current study four markers viz., CEDG006, CEDG044, CEDG097and CEDG271 were also mapped onto the single linkage group similar to Gupta et al., (2008) using the F2 population of the cross PU31 x PBG32. Chaitieng et al., (2006) constructed a genetic linkage map of blackgram using JoinMap ver. 3 at LOD score of 3.0, which has a length of 783 cM. The linked markers CEDG006, CEDG008, CEDG020, CEDG044, CEDG048, CEDG097, CEDG180, CEDG271 and MB170 that identified in the current study were also reported by Chaitieng et al., (2006). Hence, based on the current study and also from Gupta et al., (2008) and Chaitieng et al., (2006) reports, it can be assumed that the marker regions of CEDG006, CEDG044, CEDG097 and CEDG271 are highly variable across blackgram genomes warranting detailed mapping study with more number of markers.
 
QTL analysis
 
Single QTL, ‘qYMV1’for YMV tolerance was detected by using CIM that located between the markers CEDG097 and CEDG172 with LOD score value of 2.76. The phenotypic variation explained (PVE) by qYMV1 is 13.10%, which denotes a major QTL (Table 1) and the YMV tolerance allele comes from the resistant parent, PU31. The QTL is located very closer (0 cM) to the left flanking marker CEDG97, thus the marker can be used to screen YMV resistance/susceptibility among blackgram genotypes. Ver few reports are there on QTL mapping studies in blackgram. Among them, Ratanakorn et al., (2013) detected five QTLs resistant to MYMIV in India and Pakistan, the QTL qYMIV1 (CEDG100-cp02662), qYMIV2 (DMB-SSR008-VR113) and qYMIV3 (CEDG166-CEDG304)were showed resistant to MYMIV in India and the QTLs qYMIV4 (CEDG100-cp02662) and qYMIV5 (CEDG121-CEDG191)were resistant to MYMIV in Pakistan. Similarly, Mathivathana et al., (2019) reported five QTLs resistant to MYMV in cross of Vigna radiata x Vigna umbellata, two QTLs i.e., qMYMV4-1 and qMYMV5-1 were detected in the year 2015 on chromosomes 4 and 5, respectively and also three QTLs i.e.,qMYMV4-1, qMYMV6-1 and qMYMV10-1 were detected in the year 2016 on chromosomes 4, 6 and 10, respectively. They found common QTL i.e., qMYMV4-1 between the same markers VigSNP_04_32 and VigSNP_04_36 with phenotypic variance of 20.04% and 18.97% in 2015 and 2016, respectively, hence, it was concluded that the QTL is stable and is a major QTL for resistance to MYMV. The QTL region identified in the present study can be considered as novel as it is not reported in earlier studies.


 
Validation of QTL linked markers in diverse blackgram genotypes
 
The two markers, CEDG097 and CEDG172 that flanked to qYMV1 QTL were validated in known 25 resistant and susceptible blackgram genotypes. The phenotypic screening of 25 blackgram genotypes revealed, 15 genotypes showed YMV resistant while 10 showed susceptibility (Table 2). Genotyping of the 16 resistant blackgram varieties employing CEDG097 amplified the resistant allele (allele observed in PU31) of 100 bp fragment in 10 varieties, 135bp fragment (Susceptible parent allele) was amplified in one variety (TBG104), both fragments were recorded in 3 varieties and amplification was not occurred in two varieties i.e., TBG140 and VBN4. Among the susceptible blackgram varieties, the 135 bp fragment was observed in 8 out of 9 varieties and one resistant allele (100 bp) was observed in MBG1058 (susceptible variety). The marker CEDG097 showed 91% linkage in known resistant and susceptible varieties to the YMV disease reaction, when excluding the heterozygotic loci. Similarly, genotyping of the 16 resistant blackgram varieties employing marker CEDG172 was amplified the resistant allele of the 130 bp fragment in 6 varieties, 140 bp (Susceptible allele) fragment was recorded in 4 varieties and no amplicon was observed in 3 genotypes. In the susceptible varietal group, the 140 bp fragment was recorded in 6 susceptible varieties, 130 bp fragment (Resistant allele) in 3 susceptible genotypes. The primer CEDG172 exhibited 74% linkage in known varieties to the YMV disease reaction.



The genotype TBG104 showed resistant at field screening however it showed susceptible fragment alleles 135 bp (CEDG097) and 140 bp (CEDG172) at both loci, suggest that TBG104 has different gene controlled YMV resistance. Interestingly, the resistance alleles (PU31 allele) of both markers i.e., CEDG097 and CEDG172 were recorded in VBN7, LBG922, LBG933 and TBG130-1 hence, these genotypes can be considered as potent YMV resistant varieties and also can be used as donor parents in resistance breeding programmes. Similar results were reported by Gupta et al., (2013) in blackgram by validation of 20 blackgram genotypes with 10 markers. The genotype PU31 was amplified with three markers which means PU31 carrying both resistant genes and it could be used as a donor of YMV resistance in blackgram breeding programme for incorporating YMV resistance.

From the current study viz., QTL mapping and qYMV1 validation, it can be concluded that the QTL linked markers CEDG097 and CEDG172 would be employed for identification of blackgram genotypes for YMV resistance and in marker-assisted breeding approaches. However, further in depth investigation is warranted before being employed to develop YMV resistant varieties using these YMV linked markers in marker-assisted selection. 

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