Introduction
Soybean (Glycine max (L.) Merr) is an important legume crop grown worldwide for its edible oil and protein (Kim et al., Reference Kim, Ro, Kim, Kim and Chung2012; Sugiyama et al., Reference Sugiyama, Ueda, Takase and Yazaki2015). Soybean occupies an area of 119.09 million hectares and with 348.12 million tons global production (USDA, 2018). Soybean production and productivity is adversely affected by the occurrence of a wide range of biotic and abiotic stresses (Ahuja et al., Reference Ahuja, de Vos, Bones and Hall2010; Gutierrez-Gonzalez et al., Reference Gutierrez-Gonzalez, Guttikonda, Tran, Aldrich, Zhong, Yu, Nguyen and Sleper2010; Bukhari et al., Reference Bukhari, Arshad, Azooz, Azooz and Ahmad2015; Song et al., Reference Song, Prince, Valliyodan, Joshi, Maldonado dos Santos, Wang, Lin Stamatoyannopoulos, Bailey, Noble, Livak, Schmittgen, Rozen and Skaletsky2016), under the changing climatic conditions the frequency of drought occurrence enhanced, which intern leading to low production (Ali et al., Reference Ali, Ali, Quraishi, Ahmad and Rasool2014; Foyer et al., Reference Foyer, Lam, Nguyen, Siddique, Varshney, Colmer, Cowling, Bramley, Mori, Hodgson, Cooper, Miller, Kunert, Vorster, Cullis, Ozga, Wahlqvist, Liang, Shou, Shi, Yu, Fodor, Kaiser, Wong, Valliyodan and Considine2016; Dubey et al., Reference Dubey, Malla and Khan2019). Drought is one of the major abiotic stress factor responsible for nearly 40% reduction in soybean yield (Specht et al., Reference Specht, Hume and Kumudini1999; Manavalan et al., Reference Manavalan, Guttikonda, Tran and Nguyen2009). Depending upon the intensity and stage at which drought occurs, the losses could be as high as 80% (Oya et al., Reference Oya, Alexandre, Norman, Jose, Satoshi and Osamu2004; Dias et al., Reference Dias, Borges, Viana, Mesquita, Romano, Grossi-de-Sa, Nepomuceno, Loureiro and Ferreira2012). Breeding for drought tolerant cultivars is a major challenge as drought is complex trait and molecular mechanisms associated with it at the cellular level are unclear (Sinclair, Reference Sinclair2011; Cabello et al., Reference Cabello, Lodeyro and Zurbriggen2014; Deshmukh et al., Reference Deshmukh, Sonah, Patil, Chen, Prince, Mutava, Vuong, Valliyodan and Nguyen2014; Munoz et al., Reference Munoz, Li, Ngai and Miransari2016). Recent developments in molecular marker technologies and omics have resulted in the identification of new markers, genes and protein groups which are associated with abiotic stress tolerance (Collard and Mackill, Reference Collard and Mackill2008; Wang et al., Reference Wang, Sakata and Komatsu2018). Many earlier reports have indicated that drought tolerance is inherited in a simple mendelian ratio (Morgan, Reference Morgan1991; Monneyeux and Belhassen, Reference Monneyeux and Belhassen1996; Tomar and Kumar, Reference Tomar and Kumar2004). Bulked segregant analysis (BSA) techniques have been reported to be employed in the detection of large effects of genes on qualitative traits like disease resistance, wherein resistance is controlled by one or few major genes (Michelmore et al., Reference Michelmore, Paran and Kesseli1991). This method has been utilized in the detection of quantitative traits in several crops like rice (Venuprasad et al., Reference Venuprasad, Dalid, Del Valle, Zhao, Espiritu and Sta Cruz2009; Kanagaraj et al., Reference Kanagaraj, Prince, Sheeba, Biji, Paul, Senthil and Babu2010; Salunkhe et al., Reference Salunkhe, Poornima, Prince, Kanagaraj, Sheeba, Amudha, Suji, Senthil and Babu2011; Vikram et al., Reference Vikram, Swamy, Dixit, Sta Cruz, Ahmed, Singh and Kumar2011, Reference Vikram, Kumar, Singh, Singh, Tuteja, Gill, Tiburico and Tuteja2012), wheat (Altinkut and Gozukirmizi, Reference Altinkut and Gozukirmizi2003), maize (Quarrie et al., Reference Quarrie, Lazic-jancic, Kovacevic, Steed and Pekic1999) and salt tolerance in Egyptian cotton (El-Kadi et al., Reference El-Kadi, Afiah, Aly and Badran2006). Soybean plants at the seeding stage especially at the first trifoliate leaf stage is more sensitive to drought stress (www.plantstress.com) and could cause great damage to the initial establishment and survival of the crop itself. The effect of drought stress at the seedling stage is evaluated based on the seedling survival percentage and a drought score is assigned accordingly under controlled hydroponics conditions (Singh et al., Reference Singh, Dikshit and Singh2013) (Fig. 1). This was successfully demonstrated in lentil and the results perfectly correlated under pot culture experiments (Singh et al., Reference Singh, Dikshit and Singh2013). There are no reports on the seedling survivability based drought tolerance studies in soybean. Mapping for drought tolerance has been accomplished in other legumes such as mungbean (Sholihin and Hautea, Reference Sholihin2002), cowpea (Muchero et al., Reference Muchero, Ehlers, Close and Roberts2009), chickpea (Nayak et al., Reference Nayak, Zhu and Varghese2010) and lentil (Singh et al., Reference Singh, Dikshit and Singh2013). Understanding the genetic control of seedling survivability based drought tolerance in soybean would defiantly hasten the development of drought tolerant varieties in soybean.
Sequential steps in screening of soybean seedlings for drought tolerance under hydroponics conditions. 1. 
(Tolerant plants) and 2. 
(Susceptible plants).
Material and methods
Plant material
The experimental material for the present study consists of two tolerant (PK 1180 and SL 46) and two susceptible (UPSL 298 and PK 1169) germplasm lines screened for seedling survivability and drought score at the hydroponics facility at the National Phytotron Facility at ICAR-Indian Agricultural Research Institute, New Delhi. Our study concentrated mainly on seedling survivability under drought stress. The tolerant lines (PK 1180 and SL 46) were crossed with susceptible ones (UPSL 298 and PK 1169) and the F 1 plants were produced. The F 1 plants were selfed by bagging individual plants under controlled polyhouse conditions to produce F 2 seeds. The F 2 seedlings were raised in pot culture until the first trifoliate leaf stage (V3 stage) and used for screening in hydroponics and marker analysis studies.
Screening parents and F 2 population for survivability under hydroponics
The drought response of the parents and F 2 seedlings were screened under hydroponics set-up at the first trifoliate stage at the National Phytotron Facility, ICAR-Indian Agricultural Research Institute, New Delhi. Two F 2 populations from crosses PK 1180 × UPSL 298 and PK 1169 × SL 46, consisting of 126 and 82 plants respectively, were screened for seedling survivability and drought scores under in-vitro conditions. The screening method employed here is standardized in lentil by Singh et al., Reference Singh, Dikshit and Singh2013. The procedure involves, first trifoliate leaf stage seedlings were suspended in a hydroponics set-up in such a way that roots were immersed in solution. The seedlings were given drought treatment by exposing their roots to air for 4 h and 30 min for 6 consecutive days with 2 d of pre- and post-acclimatization periods. During this screening period, pH of the solution is maintained at 6.5 with 1 M of HCl or 1 M of KOH and the solution is aerated by pumping air with an aquarium air pump. At the end of screening period all the lines were rated for drought tolerance on a 0–4 scale: 0 = healthy plant with no visible symptoms of drought stress, 1 = green plants with slightly wilting, 2 = leaves turning yellowish-green with moderate wilting, 3 = leaves yellow to brown with severe wilting and 4 = completely dried leaves and/or stems. The lines with the lowest and highest drought scores were considered as tolerant and susceptible to drought stress respectively. Growth traits like root and shoot length, fresh and dry weight of root and shoot were recorded for determining drought tolerance of germplasm lines.
Data analysis
The F 2 individuals were scored as tolerant and susceptible and were subjected to the Chi-square (χ 2) test for goodness of fit to test the deviation of the observed segregation data from the theoretically expected Mendelian segregation ratio using, χ 2 = (O–E)2/E, where, O = observed number of individuals, E = expected number of individuals.
DNA extraction and SSR marker analysis
Genomic deoxyribonucleic acid (DNA) was extracted from the young leaves of the parents and F 2 seedlings at the first trifoliate stage using the cetyl trimethyl ammonium bromide procedure as described by Saghai-Maroof et al. (Reference Saghai-Maroof, Soliman, Jorgensen and Allard1984). DNA quantity was estimated by the spectrometric method at an absorbance of 260°/280°. DNA quality check was done on gel electrophoresis using 1% agarose. Polymerase chain reaction (PCR) amplification of sample DNA was performed in a thermocycler (Applied Biosystem geneamp PCR system 9700). The PCR product obtained was loaded on a 3% metaphor gel (Himedia, India) with 100 bp ladder as reference. The parental polymorphism survey was done using simple-sequence repeat (SSR) markers selected randomly from soybase (www.soybase.org). The markers are selected in such a way that entire genome is represented uniformly.
Bulked segregant analysis (BSA) and validation
The parental polymorphism survey of tolerant (PK 1180 and SL 46) and susceptible (UPSL 298 and PK 1169) genotypes was performed with the help of 368 SSR markers. The markers were selected in such a way that at least five markers should be distributed on each chromosome. The markers, which were polymorphic between the contrasting parents, were used to screen tolerant and susceptible bulks prepared from pooling of DNA from 10 tolerant and 10 susceptible F 2 genotypes, respectively. Marker linkage analysis was done using MAPMAKER version 3.0b.
Results
Inheritance of seedling survival drought tolerance trait
The crosses were made between tolerant (PK 1180 and SL 46) and susceptible (UPSL 298 and PK 1169) genotypes. In the cross PK 1180 (T) × UPSL 298 (S), 36 F 1 seeds were obtained. During the next season, F 1 plants were raised under normal conditions and were selfed to produce F 2 seeds. All of the 126 F 2s were screened for tolerance under a hydroponic assay based on seedling survivability. Of the 126, 92 genotypes showed a tolerant reaction and the remaining 34 genotypes showed a susceptible reaction under hydroponics screening conditions (Fig. 2, Table 1). In another cross PK 1169 (S) × SL 46 (T), 34 F 1 plants were selfed to produce 82 F 2 plants under controlled conditions. The F 2 population was screened for seedling survival drought tolerance under hydroponics and 60 genotypes were identified as tolerant and 22 as susceptible. The segregation analysis of seedling survival drought tolerance in the F 2 population was shown to have a goodness of fit ratio of 3 tolerant: 1 susceptible, with a probability of 0.61 at a χ 2 (3:1) of 0.258 for the cross PK 1180 × UPSL 298 and with a probability of 0.70 at a χ 2 (3:1) value of 0.145 was obtained for the cross PK 1169 × SL 46 (Table 1).
Hydroponic screening of F 2 seedlings of a cross PK 1180 × UPSL 298 for drought tolerance: a. 
Tolerant plants and b. 
Susceptible plants.
Phenotypic segregation analyses for seedling drought tolerance in F 2 population under hydroponics conditions from the crosses PK 1180 × UPSL 298 and PK 1169 × SL 46
Mapping of seedling survival drought tolerance trait
A total of 368 SSR markers were scored on tolerant (PK 1180 and SL 46) and susceptible (UPSL 298 and PK 1169) genotypes for seedling survival drought tolerance. Markers showing polymorphism between the contrasting parents were selected for mapping of drought tolerance loci in an F 2 derived mapping population (online Supplementary Fig. S1). In the process of gene mapping, we identified 36 (23%) and 43 (26%) markers that were found to be polymorphic on the genotypes used as parents in PK 1180 × UPSL 298 and PK 1169 × SL 46 cross combinations respectively (online Supplementary Table S1). The 36 markers, which had showed polymorphism for a cross combination PK 1180 × UPSL 298 were scored on tolerant, susceptible bulks and on both the parents. The primer, Satt277 gave polymorphism on both parents, the bulks and the F 2 population (online Supplementary Fig. S2). The F 2 population was grouped into three categories as homozygous tolerant, heterozygous tolerant and homozygote susceptible. The segregation analysis with marker Satt277 has produced ~100 bp susceptible allele in 31 genotypes and ~140 bp tolerant allele in 32 genotypes and the remaining 61 genotypes had both the alleles present (Fig. 3). Genetic analysis of marker segregation in the population using the χ 2 test of goodness of fit has shown a 1:2:1 ratio for a single gene with a χ 2 (1:2:1) value of 0.015 and a P value of 0.992 (Table 2). The F 2 population from the cross PK 1169 × SL 46 had 82 genotypes and it was analysed with Satt277 marked, which was found polymorphic on parents, bulks as well as segregating population. The marker produced ~100 bp susceptible allele in 17 genotypes, ~140 bp tolerant allele in 21 genotypes and the left out 44 genotypes produced both the alleles (Table 2, online Supplementary Fig. S3). Genetic analysis of marker segregation using the χ 2 test had produced 1:2:1 ratio with a χ 2 (1:2:1) value of 0.14 and a P value of 0.703. The linkage analysis of the marker using MAPMAKER indicated that the marker Satt277 is linked to seedling survival drought tolerance and is located at a distance of 3.4 cm (Fig. 4 and online Supplementary Fig. S4).
Molecular screening of F 2 population for seedling survival drought tolerance under hydroponics conditions in a cross PK 1180 × UPSL 298 with polymorphic SSR marker Satt277.L, ladder; P1, susceptible parent (UPSL 298); P2, tolerant parent (PK 1180); SB, susceptible bulk (UPSL 298); TB, tolerant bulk (PK 1180); T, tolerant reaction and S, susceptible reaction.
Genetic map of drought tolerance gene combining SSR marker on chromosome 6 in population of cross PK 1180 × UPSL 298. Tol locus, Tolerant locus.
Segregation analyses for molecular marker Satt277 closely linked to seedling drought tolerance in a F 2 population from the cross PK 1180 × UPSL 298 and PK 1169 × SL 46
Discussion
Drought stress severely reduces harvestable yield by hindering growth and metabolism in crop plants. Seedling survivability and drought score traits are considered more dependable screening methods for drought stress especially at the stage of crop. Singh et al. (Reference Singh, Mai-Kodomi and Terao1999) screened cowpea germplasm lines under artificially created drought stress conditions based on seedling survivability to differentiate drought tolerant and susceptible lines accurately. Similarly seedling survival-based screening techniques as a basis for evaluating for drought tolerance has been used in several crops such as cotton and wheat (Tomar and Kumar, Reference Tomar and Kumar2004; Basal et al., Reference Basal, Smith, Thaxton and Hemphill2005; Longenverger et al., Reference Longenverger, Smith, Thaxton and McMichael2006; Hameed et al., Reference Hameed, Goher and Iqbal2010) and lentil (Singh et al., Reference Singh, Dikshit and Singh2013). In the present study tolerant (PK 1180 and SL 46) and susceptible (UPSL 298 and PK 1169) lines were identified under hydroponics screening techniques as described by Singh et al. (Reference Singh, Dikshit and Singh2013). The contrasting parents (PK 1180 × UPSL 298) and (PK 1169 × SL 46) were crossed to develop mapping populations for drought tolerance. The mapping population showed either tolerant or susceptible reaction similar to that of the tolerant or sensitive parent when they were screened for seedling drought tolerance under hydroponics. F 2 segregation data of the mapping population obtained from crosses (PK 1180 × UPSL 298 and PK 1169 × SL 46) well fit into the χ 2 test with 3:1 ratio of tolerance over susceptibility confirmed single major gene involved in early drought/seedling drought tolerance.
In the present study, BSA has been effectively utilized in the identification of the SSR marker, tolerant to early or seedling drought tolerance. The marker, Satt277 identified as polymorphic between tolerant and sensitive bulks was mapped with the seedling survival drought tolerance using single marker analysis. The makers is co-segregating with trait and it is located on a chromosome linkage group C2 (chr 6). Several examples of BSA are being utilized in mapping the complex abiotic stresses like salinity, drought, waterlogging and extreme temperatures as well as quantitative trait loci (QTL's) in many crops are available (Venuprasad et al., Reference Venuprasad, Dalid, Del Valle, Zhao, Espiritu and Sta Cruz2009; Kanagaraj et al., Reference Kanagaraj, Prince, Sheeba, Biji, Paul, Senthil and Babu2010; Salunkhe et al., Reference Salunkhe, Poornima, Prince, Kanagaraj, Sheeba, Amudha, Suji, Senthil and Babu2011; Vikram et al., Reference Vikram, Kumar, Singh, Singh, Tuteja, Gill, Tiburico and Tuteja2012). BSA is more cost-effective than the selective genotyping (SG) method to study association between markers and traits, where less information is available regarding their inheritance and also comparing bulk samples is easier than evaluating individuals in different populations as is done in conventional linkage mapping (Singh et al., Reference Singh, Singh, Taunk and Tomar2016). Altinkut and Gozukirmizi (Reference Altinkut and Gozukirmizi2003) used BSA to identify microsatellite markers that are associated with water stress in wheat. Venuprasad et al. (Reference Venuprasad, Dalid, Del Valle, Zhao, Espiritu and Sta Cruz2009) identified markers linked to two loci associated with grain yield under lowland drought stress in rice crop. Wang et al., Reference Wang, Sakata and Komatsu2018 reported that the protein levels of glyceraldehyde-3-phosphate dehydrogenase, aconitase-1 and 2-oxoglutarate dehydrogenase were higher under stress conditions and might play an essential role in conferring tolerance in soybean. In another study, Vikram et al. (Reference Vikram, Kumar, Singh, Singh, Tuteja, Gill, Tiburico and Tuteja2012) were identified QTL's associated with grain yield in rice under drought stress using the BSA method and its effectiveness is confirmed with SG and whole population genotyping. Salunkhe et al. (Reference Salunkhe, Poornima, Prince, Kanagaraj, Sheeba, Amudha, Suji, Senthil and Babu2011) were fine mapped the drought resistant QTL on a chromosome 1 in rice using BSA. Ullah et al. (Reference Ullah, Jun and Kyong2018) in maize identified major alleles responsible for drought resistance and susceptible traits using recombinant inbred line (RIL) population. In another study, Kanagaraj et al. (Reference Kanagaraj, Prince, Sheeba, Biji, Paul, Senthil and Babu2010) identified SSR markers associated with drought resistance component traits in RIL populations from a cross between IR 20 × Nootripathu using BSA in rice. BSA was carried in soybean for different parameters like soybean rust resistance (Hyten et al., Reference Hyten, Smith, Frederick, Tucker, Song and Cregan2009) and flowering time (Watanabe et al., Reference Watanabe, Chikaharu, Oshita, Yamada, Anai and Kaga2017). Simple inheritance of seedling survival drought tolerance is probably due to the fact that seedling ability to recover fully and continue normal metabolism after sudden shock treatments (drought exposure under hydroponics) is otherwise known as resurrection tolerance, which is the main focus of the present study. Other traits such as root and physiological traits contributions were minimal. Further, at the molecular level, the monogenic nature of seedling survival drought tolerance is due to the involvement of several regulatory factors performing the same function. There are many earlier studies reported that drought tolerance is inherited in a simple Mendelian pattern in several crops. Morgan (Reference Morgan1991) reported that osmoregulation in response to water stress in wheat is controlled by a single recessive gene. Tomar and Kumar, (Reference Tomar and Kumar2004) have concluded that inheritance of seedling survivability based drought tolerance is controlled by a single dominant gene in wheat. Similar results were available in cotton (Monneyeux and Belhassen, Reference Monneyeux and Belhassen1996) and in lentil (Singh et al., Reference Singh, Dikshit and Singh2013). The present study helped in identifying drought tolerant and susceptible genotypes based on seedling survivability when screened under hydroponics conditions. The genotypes identified as tolerant for seedlings survival drought tolerance could be used as a donor and the marker identified (Satt277) can be used in marker assisted selection after validation, for transfer of the trait to other drought sensitive varieties of soybean. The marker can also be effectively utilized on a large scale screening of germplasm lines for drought tolerance.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262120000052.
Acknowledgements
The authors are highly thankful to Director, ICAR-IARI, New Delhi for providing facilities and support. The authors acknowledge the support of The Head, Genetics Division, ICAR-IARI New Delhi. The first author is highly thankful to Director, ICAR-SBI, Coimbatore for allowing an In-Service Ph.D at ICAR-IARI, New Delhi.
Conflict of interest
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript and in the decision to publish the results.
