High‐throughput NGS‐based genotyping and phenotyping: Role in genomics‐assisted breeding for soybean improvement

Soybean is an important food crop that provides edible protein and oil for human and animal nutrition. Conventional phenotypic‐based breeding approaches have made significant contribution in the last century by developing many improved soybean varieties. However, due to the longer time taken to develop a variety, low genetic gain per unit time, and adverse environmental influence of phenotypic‐based selection, conventional approaches are not sufficient to maintain pace with growing population and climate change. In this context, the recent method of genotypic selection, that is, genomics‐assisted breeding (GAB) is considered a promising approach to address the challenges in soybean breeding. However, to harness the true potential of GAB in soybean improvement, the great coverage and precision in the genotyping and phenotyping are required. Previously, a huge gap was observed between the discovery and practical use of quantitative trait loci (QTLs) in soybean improvement. It has been suggested that low marker density and manually collected phenotypes are the major reasons for this failure. Hence, high‐throughput genotyping (HTG) providing higher genome‐wide marker density, as well as accurate and precise phenotyping using high‐throughput digital phenotyping (HTP) platforms, can significantly increase the success of QTL and candidate gene identification in soybean. These approaches can greatly increase the practical utility of GAB in soybean and also offer a faster characterization of germplasm and breeding materials. This review provides the detailed information on how the recent innovations in genomics and phenomics can assists in improving the efficiency and potential of GAB in soybean improvement.


| INTRODUCTION
Soybean (Glycine max L. Merr.), being a rich source of edible oil and protein, is an economically important crop. It provides significant benefits in health, biofuel, and soil fertility improvement (Kulkarni et al., 2016). In China, soybean production has continuously declined due to lower yield increases in the past 50 years (Liu et al., 2018).
China imports >80% of soybean for their total domestic use; hence, it is essential to increase the domestic production of soybean to make the country self-sufficient (Liu et al., 2018). Soybean production can be increased mainly by breeding high-yielding stress-tolerant cultivars . In this regard, the different yield-related traits are targeted by plant breeders to increase soybean production (Karikari et al., 2019). In the last century, conventional breeding approaches have tremendously contributed by successfully developing many high-yielding and superior quality soybean varieties (Ahmar et al., 2020). However, the progress achieved through the conventional approaches are not sufficient to keep pace with growing population and climate change (Athar & Ashraf, 2009;Bhat et al., 2016). As most of the economically important traits in soybean such as yield, yield-related traits, and stress tolerance are complex quantitative traits (Karikari et al., 2019;Hina et al., 2020 ), the time-consuming and expensive conventional methods employed for the phenotypic selection of the desired plant are not sufficient to maintain global food security (Bhat et al., 2016;Mir et al., 2019).
The recent progress made in genomics and phenomics provides exciting opportunities to conduct precision breeding at higher efficiency (Mir et al., 2019;van Bezouw et al., 2019). For example, the genomics-assisted breeding (GAB) have allowed more genetic gain for complex traits at a relatively lower cost, but it requires genomic resources and molecular understanding of the trait (Chaudhary et al., 2015;Steiner et al., 2019). GAB involves two main important approaches, namely, marker-assisted selection (MAS) and genomic selection (GS) (Bhat et al., 2016). MAS depends upon the availability of markers associated with the trait of interest which can be identified either through linkage mapping or genome-wide association studies (GWAS). Many previous studies have demonstrated successful utilization of MAS in soybean by incorporating major genes and large-effect quantitative trait loci (QTLs) for different traits (Concibido et al., 1996;Gupta et al., 2017;Saghai Maroof et al., 2008). However, the minor genes govern most of the inheritance in the complex traits, but they have been never considered because of the limitations of MAS (Spindel et al., 2015). Further, the environment influence, epistatic interactions, and effect of genetic background have made the breeding of complex traits very complicated. Therefore, plant breeders have realized that MAS is not a right approach to breed complex plant traits. In this context, Meuwissen and Goddard (2001) have put forward the concept of GS as an alternative strategy.
In contrast to traditional MAS, GS utilizes whole genome-wide marker profile of breeding lines to predict the genomics-estimated breeding value (GEBV) by using different models; thus, it prevents the loss of the substantial portion of variation governed by minor effect QTLs/genes (Spindel et al., 2015). However, the accuracy in the identification of marker-trait associations and estimation of GEBV depends upon precise genotyping and phenotyping analysis, which in turn determine the success of GAB. The manual low-throughput phenotyping and genotyping often lead to false positive or negatives selection (Tuberosa, 2012). In this regard, high-throughput next generation sequencing (NGS)-based genotyping and phenotyping allow effective MAS and GS, and offer increased success of breeding programs (Cobb et al., 2019;Khan et al., 2016). Availability of highthroughput NGS-based genotyping techniques has considerably accelerated the process of gene discovery and GS, that too in crop plants with larger and complex genomes such as soybean (Bhat et al., 2016).
To this effect, phenomics and genomics are equally critical for precise gene discovery and deriving GS model to estimate the GEBV of the breeding population (BP). Therefore, combining these approaches with appropriate genetic diversity, soil and weather data, analytical F I G U R E 1 Diagram showing the critical role of high-throughput phenotyping (HTP) and high-throughput genotyping (HTG) in the precise and accurate identification of quantitative trait loci (QTLs)/quantitative trait nucleotides (QTNs)/genes as well as genomic selection (GS)-based estimation of genomics-estimated breeding values (GEBVs) tools, and databases would result in development of new varieties with enhanced yield, quality, and stress tolerance at a fast pace.
In the present review, we provide detailed information on how the advances in the high-throughput phenotyping (HTP) and highthroughput genotyping (HTG) approaches will help in increasing the precision of gene mapping and genomic prediction. We also discuss how these techniques are going to prove fruitful for developing nextgeneration crop improvement strategies in soybean and other crop species.
2 | GENOMICS-ASSISTED BREEDING GAB involves two main approaches, namely, MAS and GS (Figure 1; Bhat et al., 2016). The MAS (includes MABC and MARS) utilizes molecular markers known to be associated with a particular trait/phenotype to identify a desirable individual carrying favorable allele for the trait of interest (Jiang, 2013). However, MAS is applicable only for the major-effect genes (Bhat et al., 2016); for example, most of the successful MAS-based studies carried out earlier in soybean involved mainly the major-effect QTLs/genes that govern large portion of phenotypic variation for the trait of interest (see details in Concibido et al., 1996;Gupta et al., 2017;Kim et al., 2020;Saghai Maroof et al., 2008;Viganó et al., 2018). In case of minor genes, which contribute only a small invisible phenotypic variation for the complex trait, MAS has not led to successful results in soybean (Bhat et al., 2016;Zhang et al., 2015). Most of the economically important traits in soybean such as yield, oil and protein content, and the stress tolerance are complex in nature; the maximum portion of phenotyping variation for these traits are controlled by minor effect genes (Hina et al., 2020). In this regard, GS offers promising results for complex quantitative traits (Figure 2; Bhat et al., 2016;Stewart-Brown et al., 2019). The GS combines marker profile and phenotypic data of the training population to develop the prediction model, and this model is used to estimate the GEBV of all individuals of BP (Gao et al., 2018;Yin et al., 2020). However, prior to the use of prediction model for the selection of individuals from BP, the cross-validation on subsets of the training population are used to check the accuracy of this model (Bhat et al., 2016). After successful validation, this model can be used to select individuals from the BP based on the GEBVs calculated only from the marker/genotypic data; hence, only the genotypic information is used to predict the phenotypic performance of individuals of the BP (Stewart-Brown et al., 2019). Recently, some of the GS studies carried out in soybean using high-throughput single nucleotide polymorphism (SNP) genotyping have revealed promising results for different complex traits with moderate to high prediction accuracy ( Table 1).
The major advantage of GAB is the genotypic data collected from an early plant growth stage (such as seedling) can be used to predict the phenotypic performance of mature individuals. Thus, it can considerably save time, money, and labor needed for the extensive phenoDordevictypic evaluation in multiple environments and years Kole et al., 2015). In addition, GAB allows a greater number of breeding selection cycles and genetic gain per unit time (Bhat et al., 2016). In the past few decades, the complete reliance on phenotypic selection has steadily changed to more increased use of genotypic-based methods for plant selection, especially made possible by NGS-based genotyping platforms (Jarquín et al., 2014;Matei et al., 2018;Qin et al., 2019;Ravelombola et al., 2020). The NGS technologies have enabled increased throughput, cost-effectiveness, and speed of genome-wide genotyping (Getachew, 2019). Before the emergence of NGS-based marker genotyping, the development of F I G U R E 2 The role of NGS-based marker genotyping and high-throughput phenotyping (HTP) on the success of genomic selection (GS) in crop/soybean breeding. The NGS and HTP offers promising results of GS in the development of improved soybean varieties in less period of time span markers was expensive and laborious, and GAB, especially the GS, was constrained by the number of markers that could efficiently be assayed (Bhat et al., 2016). As a result, only markers in critical genomic regions were utilized to predict the presence or absence of agriculturally valuable traits (Varshney et al., 2014). Both GAB approaches, namely, MAS and GS, have different limitations and strengths, and use of either approaches in soybean breeding depends upon the trait architecture and heritability as discussed above (Mir et al., 2019;Varshney et al., 2014).

| WHOLE GENOME RESEQUENCING
Whole genome resequencing (WGRS) is the ultimate approach to detect all available genetic variations/polymorphism present in a BP of a particular crop species. However, the use of WGRS in marker genotyping requires the availability of high-quality reference genome sequence for a particular crop species. In this context, since the publication of the first high-quality soybean reference genome (Schmutz et al., 2010), the follow-up WGRS studies have allowed the whole genome investigations in soybean (Zhou et al., 2015). The comparison of the resequencing information generated from the BP with the high-quality reference genome has led to the identification of all kinds of genetic variation present in genetic populations. This approach has the advantage of providing potential access to whole genome level variations, such as SNPs, insertions, deletions, transversions, and copy number variants (Zhou et al., 2015). WGRS has increased the resolution of gene mapping and candidate gene identification. This has ultimately increased the applicability and success of GAB in soybean improvement ( Figure 1; Jaganathan et al., 2020).
Among the various NGS-based marker genotyping approaches, the WGRS approach is the only approach to detect all genome-wide polymorphisms for their ultimate use in GAB (Bhat et al., 2016). The WGRS will provide highest marker density and resolution of gene mapping, as well as precise estimation of GEBVs; hence, among all the NGS genotyping methods, the WGRS will provide the maximum benefits from genomic revolution in crop improvement (Zhou et al., 2015).
However, it has some important limitation especially in the large and complex genomes such as soybean that prevents its routine commercial use in the soybean breeding programs. These limitations include high sequencing costs (long reads and chromosome scale scaffolding techniques are needed), requirement of complex data analysis, and demand for high-performance computing platforms. The continuous decline in sequencing cost will make use of WGRS feasible and cost effective in different applications of soybean breeding in the near future. Until then, the reduced representation sequencing especially T A B L E 1 Genomic selection (GS) studies for different quantitative traits in soybean using high-throughput SNP genotyping platforms T A B L E 2 Studies related to GS, GWAS, and linkage mapping involving the use of NGS-based high-throughput genotyping for the identification of marker-trait associations and prediction accuracy in soybean Glyma.08g146100, Glyma.08g224400, Glyma.02g204300, Glyma.14g211300, and Glyma.08g157400   the genotyping by sequencing (GBS) seems to be more cost-effective option for large-scale marker discovery and GS (Tables 1 and 2; Bhat et al., 2016;Nepolean et al., 2018).
Efforts have been made in soybean utilizing the WGRS for genotyping and marker generation in gene mapping and domestication studies (Table 2) production procedure more amenable to use on large numbers of individuals/lines (Elshire et al., 2011;Poland et al., 2012).
Recently, reduced cost of GBS has made it increasingly more feasible in soybean compared with WGRS as a method of genotyping (Tables 1 and 2; Alipour et al., 2019;Fang et al., 2019;Gutierrez-Gonzalez et al., 2019;Jiang et al., 2020). GBS has been routinely used in the gene mapping and other studies in soybean (Table 2). GBS has been demonstrated to be an efficient technique for high-resolution genetic mapping and precision breeding in soybean (Getachew, 2019).
Recently, GBS has been used to sequence collections of different mapping population (such as RIL and F 2 ) and diversity panels, to analyze and map various traits in soybean (Table 2). GBS has considerably facilitated the high-resolution gene mapping and candidate gene identification in soybean (Fang et al., 2019;Sonah et al., 2015;Wang et al., 2020). GBS have also been used for de novo genotyping of soybean breeding panels and to develop accurate GS models ( in the prediction accuracy for GEBV has been compared with arraybased and SSR marker systems (Heslot et al., 2013). Through intensive multiplexing, this approach can reduce the cost to below 10 dollars per sample (Bhat et al., 2016). The GBS cost per individual is much lower compared with array-based and other NGS-based markers in soybean (Bassi et al., 2016;Getachew, 2019). Moreover, GBS covers much higher fraction of the genome compared with even the densest SNP arrays currently available in soybean plant (Bhat et al., 2016;Gorjanc et al., 2015). The SNP arrays are typically developed from a limited sample of individuals, whereas GBS can capture genetic variation that is specific to a population or family of interest. GBS has the advantage that markers are discovered using the population to be genotyped, thus minimizing ascertainment bias. The flexibility, low cost, and GEBV prediction accuracy of GBS make this an ideal approach for GAB applications in soybean in the current era (Bhat et al., 2016).

| NGS-BASED GENOTYPING: GENE DISCOVERY AND MAS IN SOYBEAN
Prior to the use of MAS approach in crop breeding, it is a prerequisite to have initial knowledge of the major-effect gene/QTL serving as a target in the selection process. In this regard, the NGS-based genotyping technologies have greatly benefited in increasing the resolution of gene mapping and tagged the gene/QTL very close to the adjacent maker (Figure 1). For example, in the GWAS analysis, the use of NGS has made it possible to genotype large populations of plants with a higher density of markers that was previously impossible, and this contributes directly to increased mapping resolution (Jing et al., 2018;Li et al., 2019a). Use of diverse and large population in GWAS analysis allows the detection of more recombination break points that in turn help in the identification of candidate genes at higher precision (Alqudah et al., 2020). Many studies have utilized NGS-based genotyping for GWAS analysis in soybean for the different traits; and these studies have revealed considerable success in the candidate gene identification for particular traits of interest (see details in Table 2 (Table 2). It has been demonstrated that NGS also possesses great capability to resolve the genome duplication issues in a complex and paleopolyploid soybean crop Xu et al. (2013).
MAS is an earliest form of GAB and has been extensively used for the breeding of improved crop varieties (Cobb et al., 2019). Application of MAS in crop breeding needs a marker tagged to a particular gene/QTL regulating the phenotypic variation for the trait of interest (Collard & Mackill, 2008). Trait-linked marker is used in MAS, which involves the introgression of tagged gene/QTL into elite background of superior crop variety. As discussed in detail above, high-throughput SNP genotyping has greatly increased the resolution of gene tagging, thereby significantly increasing the success of MAS (Figure 1). HTG has been successfully used to introgress target loci into elite varieties to improve their performance for specific trait (Jaganathan et al., 2020;Varshney et al., 2014). Patil et al. (2016) developed a high-throughput SNP-marker assay for MAS of salinity tolerance in soybean. Similarly, Shi et al. (2015) have generated a SNP-marker assay for high-throughput selection of soybean cyst nematode (SCN), and they demonstrated that the use of one or two of these markers is sufficient for high-throughput MAS of soybean plants showing enhanced SCN resistance. In addition, the NGS-based genotyping has considerably reduced the linkage drag involved in the MAB (Varshney et al., 2014). Breeding program involved in the transfer of favorable allele/gene via MAB from the wild unadapted parent into the elite cultivated recipient parent often leads to linkage drag (Mir et al., 2014).
Breeders do not want this linkage drag, that is, transfer of undesirable genes linked to desirable gene into the elite recipient parent, because they often make the new developed cultivar unacceptable to the farmers and consumers (Mir et al., 2014). In this regard, NGS has

| NGS-BASED GENOTYPING VERSUS SUCCESS OF GS IN SOYBEAN
Sanger sequencing was the initial technique used for the whole genome sequencing (WGS) of model crop plants (Edwards & Batley, 2010). Availability of WGS has increased the scope of sequence-based markers such as SSR and SNP and has greatly revolutionized the marker technology in plants (Zargar et al., 2015). But many factors such as high cost, more time, and information restricted to target individual has limited the use of Sanger technology for specific gene discovery (Ray & Satya, 2014). Hence, this sequencing technology is not feasible for commercial use either in small or large breeding programs (Bhat et al., 2016). The recent advances in the NGS technologies and powerful computational pipelines has reduced the cost of WGS/WGRS to great extent allowing the discovery, sequencing, and genotyping of hundreds of thousands of markers in a single step (Stapley et al., 2010). As discussed in the previous section, NGS-based WGRS is not currently commercially feasible for large breeding programs due to its higher cost, but in the near future, the cost for resequencing a genome will decrease to only a few hundred US dollars. Until then, the NGS-marker technologies based on reduced representation sequencing is the best option for large-scale marker discovery, especially for the large and complex soybean genome.
These NGS-based marker techniques represent the partial genome of the soybean, and they even can be performed in the absence of a reference sequence (Ray & Satya, 2014;Toonen et al., 2013). Among these NGS technologies, RAD-seq (or its variants) and GBS have already proven to be efficient and effective techniques for GAB and were frequently used for GS studies in different crop plants (Tables 1   and 2; Glaubitz et al., 2014;Yang et al., 2012). In addition, the NGS has allowed the development of high-density SNP chips for conducting HTG in soybean (Table 1).
Multiple features of NGS-marker technologies, namely, low cost, genome-wide marker coverage, higher speed and throughput, and higher marker density have allowed the geneticist to study the inheritance of many traits at the nucleotide level precision (Chung et al., 2017). In contrast, the previous low-throughput marker technologies (such as RFLP, AFLP, and SSR) have restricted the use of markers only in critical genomic regions such as MAS, to predict the presence or absence of agriculturally important traits (Bhat et al., 2016;Varshney et al., 2014). These marker types were not efficient and cost effective to get the better results from the GS, and thus, the GAB was limited to only MAS. However, the availability of NGS genotyping platforms has widened the scope of GAB in crop improvement leading to the great success of GS, leading to substantial shift from the phenotyping-based selection to increased reliance on genotyping-based selection (Houston et al., 2020). Compared with array-based marker systems, the NGS genotyping has offered many advantages such as lack of ascertainment bias, low genotyping cost (per sample cost < $20 USD), and higher marker density that makes them marker of choice for GS (Bhat et al., 2016;Poland et al., 2012).
In case of GS, the marker density and size of reference population are the most important factors affecting the efficiency and accuracy of GS in the prediction of superior genotypes, and both of these factors are limited by high genotyping cost Varshney et al., 2014) that are now resolved by NGS genotyping technologies (Bhat et al., 2016;Jarquín et al., 2014). Moreover, the prediction accuracy of GS is also affected by population structure that often leads to biased accuracies of genomic predictions (Stewart-Brown et al., 2019;Wray et al., 2013). NGS-based marker genotyping compared with earlier marker systems (SSR and array-based markers) provides higher genome-wide marker density to precisely determine the population structure of TP; thus, it enables accurate estimation of the GEBV of BP (Isidro et al., 2015;Qin et al., 2019).
The beneficial features of NGS technology has made the NGSbased genotyping as a cost-effective and efficient agrigenomics tool for performing GS in soybean (Kirst et al., 2011;Metzker, 2010;Poland et al., 2012;Toonen et al., 2013). GS has been attempted in soybean for grain yield traits using GBS, and genome-wide prediction accuracies were investigated to be high (average of 0.64). GS was suggested to have good potential for grain yield in soybean (Jarquín et al., 2014). Zhang et al. (2016) also demonstrated higher prediction accuracy of GS (0.75-0.87) compared with MAS (0.62-0.75) for grain weight in soybean using high-throughput SNP genotyping. Thus, GS outperforms MAS for grain weight in soybean. In addition, the GS was also demonstrated to be superior compared with phenotypic selection for multiple traits such as grain yield, plant height, insertion of first pod, days to maturity and 1000-grain weight in soybean, and selection time is reduced in 50%, that is, from 6 to 3 years (Matei et al., 2018). Besides, many studies have demonstrated that GS can reduce the time required to complete a selection cycle in crop plants, which can lead to increased production of this commercially important crop (Bhat et al., 2016;Qin et al., 2019). List of studies involving the use of high-throughput SNP-and NGS-marker genotyping for conducting GS in soybean for the different traits showing moderate to high predication accuracy are presented in Table 1. All these studies have demonstrated the great potential of GS in accelerating the soybean production and productivity. Presently, the cost of NGS and WGRS genotyping is still high to commercially use these platforms for conducting GS in a soybean (Bhat et al., 2016;Varshney et al., 2019). The high cost has limited the use of these genotyping platforms to the smaller plant population. The reduction of sequencing cost in the future will allow use of genotyping at commercial scale in the large collection of soybean germplasm stored in the genebanks to predict the phenotype at higher accuracy (Bhat et al., 2016. The NGS-marker genotyping have been documented to give higher GS accuracy than array-based markers on the same lines of crop plants, even in the case of 43.9% missing data (Heslot et al., 2013). All of these features have made the sequencebased genotyping an ideal approach for GS and its successful application in soybean breeding ( Figure 2).

| HIGH-THROUGHPUT PHENOMICS: CURRENT SNAG IN SOYBEAN BREEDING
In the past decade, the advances made in the NGS and HTG technologies have greatly improved our understanding of crop genomes. These advances have considerably increased the scope of crop breeding and genetics (Zargar et al., 2015). Sequencing of plant and crop genomes has become a routine process . Due to the advances in NGS technology, draft genome sequences of multiple legume crops including soybean have now become available (Legume Information System, https://legumeinfo.org). The allelic variation has also been identified by using WGRS in soybean (Bhat et al., 2016;Patil et al., 2019;Zhou et al., 2015). Genotyping cost has also considerably reduced due to the availability of HTG platforms in soybean (Pandey et al., 2016). However, to harness the true potential of the genomic revolution in soybean improvement, high-quality genomic data are needed to be integrated with precise and accurate phenotypic data in a real-world environment (Figures 1 and 2; Furbank & Tester, 2011).
The traditional manual phenotyping used in soybean was laborious, time consuming, destructive, and biased that often led to inaccurate results and at times resulted in failure of breeding programs (Bhat et al., 2016). The use of HTP technologies can considerably reduce the cost and labor for the phenotyping of different traits in soybean and increase the accuracy and precision in the crop breeding compared with traditional manual phenotyping (Moreira et al., 2019(Moreira et al., , 2020Singh et al., 2016;Trevisan et al., 2020). So far, very limited efforts have been made to develop high-throughput advanced phenotypic approaches for crop breeding (Mir et al., 2019). Thus, in the current era of soybean breeding research, this has become a major snag in utilizing the abundant high-quality available genomic data (www.soybase.org) that in turn affects the progress of breeding research in soybean (Edwards et al., 2013;Stamp & Visser, 2012 HTPheno (Hartmann et al., 2011) and IAP (Klukas et al., 2014); incorporating the open-source software ImageJ needs to be developed and popularized.

| PERSPECTIVES AND CONCLUSION
In the past century, conventional breeding has contributed significantly to soybean improvement and development of soybean varieties with improved yield, quality, and tolerance to multiple stresses. However, conventional methods of crop improvement are not able to maintain pace with the accelerated growing population and climate change (Gosal et al., 2009). GAB is suggested to have great potential in overcoming the future challenges in soybean improvement (Collard & Mackill, 2008). But the true potential of GAB can be harnessed only by precisely and accurately identifying the marker-trait associations (via linkage mapping and GWAS) and estimation of GEBV However, to harness real benefits from genomic studies, these marker technologies must be combined with HTP to achieve valuable genetic gain from complex traits. In soybean, very few studies involving the combined use of both HTP and HTG have been published so far. The reason for this is the higher cost involved in the large-scale field-based HTP. It has been predicted that in the near future, new innovations in crop phenotyping technology will allow HTP affordable for its commercial use in the soybean breeding programs. This will definitely expand the scale of germplasm evaluation and will allow the rapid development of improved soybean varieties. The declining cost of DNA sequencing will also make WGRS-based genotyping more feasible and cost effective. At present, for breeding-based applications such as GS, sequencing-based genotyping using genome-reduction techniques such as GBS and RAD-sequencing seems to be more cost effective.

ACKNOWLEDGMENT
This research was funded by the Ministry of Science and Technology of China (2017YFE0111000).

CONFLICT OF INTEREST
The authors declare no competing financial interests.

ETHICS STATEMENT
Not applicable.

AUTHOR CONTRIBUTIONS
Javaid Akhter Bhat and Deyue Yu have contributed equally in the development of this manuscript.

DATA AVAILABILITY STATEMENT
The data sets used during the current study are available from the corresponding author on reasonable request.