Introgression of “ QTL-hotspot ” region enhances drought tolerance and grain yield in three elite chickpea cultivars

With an aim of enhancing drought tolerance using a marker-assisted backcrossing (MABC) approach, we introgressed the “ QTL-hotspot ” region from ICC 4958 accession that harbors quantitative trait loci (QTLs) for several drought-tolerance related traits into three elite Indian chickpea ( Cicer arietinum


INTRODUCTION
Chickpea (Cicer arietinum L.) is a cool-season food legume cultivated on residual soil moisture in southern Asia and sub-Saharan Africa. Being a rich source of protein, fiber, and other mineral nutrients, it is important for global nutritional and food security. Southeastern Turkey is considered the center of origin of chickpea and after its domestication in the Middle East, chickpea has migrated to the Mediterranean region, India, and Ethiopia (Ladizinsky, 1975;van der Maesen, 1987). Further, in recent years, an increase in cultivated area (17.81 million ha) and production (17.19 million tonnes) has been evidenced (FAOSTAT, 2019). In the case of India, the largest producer and consumer, chickpea production increased from 3.86 to 11.23 million tonnes between 2000-2001(Dixit, Srivastava, & Singh, 2019. Madhya Pradesh, Maharashtra, Rajasthan, Karnataka, and Andhra Pradesh are major chickpea growing states in India. Abiotic and biotic stresses hamper chickpea production, especially terminal drought or end-season drought has alone been reported to cause >50% yield losses (Ahmad, Gaur, & Croser, 2005). Globally, the frequency of occurrence and severity of drought is predicted to increase in the climate change scenario (Carrão, Naumann, & Barbosa, 2018). In India, during the last five decades, drought has been reported to occur at least once in every 3 yr (Mishra, Singh, & Desai, 2009, United Nations Office for Disaster Risk Reduction, 2009). In the case of central and southern India, where the occurrence of drought is more frequent, to mitigate the adverse effects of drought, the chickpea research community has leveraged drought escape and drought avoidance mechanisms (Berger, Palta, & Vadez, 2016;Gaur et al., 2019). According to the Vision 2050 document of Indian Council of Agricultural Research (ICAR)-Indian Institute of Pulses Research (IIPR), about 16-17.5 million tonnes of chickpea needs to be produced by 2050 from an area of about 10.5 million ha with average productivity of 1.5-1.7 t ha −1 https://iipr.icar.gov. in/pdf/vision_250715.pdf). However, to achieve this selfsufficiency, deeper understanding of genetics of drought tolerance and developing the drought-tolerant chickpea cultivars is required. Drought being a complex trait, earlier efforts to understand the genetics of the trait had a major focus on morphological, biochemical, and physiological traits associated that contribute to drought tolerance (Gunes et al., 2006;Mafakheri, Siosemardeh, Bahramnejad, Struik, & Sohrabi, 2010;Purushothaman, Krishnamurthy, Upadhyaya, Vadez, & Varshney, 2016;Upadhyaya et al., 2012;Varshney, Tuberosa, & Tardieu, 2018). The role of traits like transpiration efficiency and carban isotope discrimination in mitigating terminal drought was also reported (Kashiwagi et al., 2005). In a recent study, conservative water use benefitting seed yield of chickpea under terminal drought conditions has been reported (Pang, Turner, Du, Colmer, & Siddique, 2017). The traits of relevance and scope for improving yield under drought has also been presented (Kashiwagi et al., 2013. Root traits, especially root length density (RLD [g cm −3 ]) was reported to play a key role in mitigating the effects of drought by studying the root system architecture and its plasticity in chickpea germplasm lines (Kashiwagi, Krishnamurthy, Gaur, Chandra, & Upadhyaya, 2008). Anatomical studies on chickpea root system showed moderate xylem passage (number of xylem vessels × average vessel diameter) per root indicating that chickpea is capable of absorbing water moderately under drought conditions (Purushothaman et al., 2013). Besides profuse RLD at surface soil depths, root dry weight at deeper soil layers and high root/shoot ratio was proposed to be the best selection strategy for yield under terminal drought conditions in chickpea (Purushothaman, Krishnamurthy, Upadhyaya, Vadez, & Varshney, 2017).

Core Ideas
• Sixty-one backcross progenies with >90% recurrent parent genome recovery were developed. • Six superior lines with enhanced drought tolerance and yield performance were nominated for national yield trials in India. • Pusa Chickpea 10216, the first molecular breeding variety for drought tolerance, was released in India.
to Fusarium wilt and Ascochyta blight have been reported using the MABC approach (Mannur et al., 2019;Pratap et al., 2017;Varshney et al., 2014b). Fusarium wilt resistant variety MABC-WR-SA1, also called Super Annigeri 1, with 7% more yield potential than Annigeri 1 (Mannur et al., 2019) has also been released for commercial cultivation in India by Central Sub-Committees on Crop Standards, Notification and Release of Varieties of Agricultural Crops, Ministry of Agriculture and Farmers Welfare, Government of India.
The present study reports the development of 61 superior lines with higher seed yield and enhanced drought tolerance in three genetic backgrounds, namely Pusa 372 and Pusa 362 at ICAR-Indian Agricultural Research Institute (IARI) and DCP 92-3 at ICAR-IIPR in collaboration with International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). We also report the release of Pusa Chickpea 10216, a molecular breeding variety with enhanced drought tolerance and increased yield under rainfed conditions in India. Our study confirms that the introgression of the QTLhotspot region enhances drought tolerance and yield irrespective of the genetic backgrounds.

Selection of donor and recipient parents
With an aim of enhancing the drought tolerance in elite cultivars, the MABC approach was adopted independently at ICAR-IARI and ICAR-IIPR. For achieving this, ICC 4958 (http://oar.icrisat.org/540/1/PMD_33.pdf) was used as donor parent to introgress the QTL-hotspot into three elite cultivars namely Pusa 372, Pusa 362, and DCP 92-3. Donor ICC 4958 is a landrace collected from Jabalpur, Madhya Pradesh, India, in 1973. It is used as a drought-tolerant donor parent that produces high yields in short-duration, terminal-drought environments regions.

Markers for foreground and background selection
Initially, eight SSR markers (TAA170, ICCM0249, GA24, STMS11, NCPGR21, NCPGR127, GA11, and TR11) in the QTL-hotspot region  were used for parental polymorphism analysis on three selected recipient parents (Pusa 362, Pusa 372, and DCP 92-3) and donor parent (ICC 4958) for possible use in foreground selection. Polymerase chain reaction (PCR) for all markers as performed in 5-μl reaction volume as described earlier (Varshney et al., 2013a). The PCR amplicons were either resolved on 1.2% agarose gel or using ABI 3730 (Applied Biosystems). For foreground selection, polymorphic markers with donor and recipient cross-combinations from the QTL-hotspot genomic region were used (Supplemental Table S1). Based on earlier studies, a set of 346 highly polymorphic SSR markers (Nayak et al., 2010;Thudi et al., 2011;Varshney et al., 2014a) were tested for parental polymorphism among donor and recipient parents for possible use in background selection at the Center of Excellence in Genomics and Systems Biology (cegsb.icrisat.org), ICRISAT. Polymorphic markers identified for each donor and recipient parent cross-combination were used for background selection (Supplemental Table S2). The percentage of recurrent parent genome (RPG) recovery was calculated as described earlier (Mannur et al., 2019).

Introgression of the QTL-hotspot genomic region into three genetic backgrounds
The QTL-hotspot region was introgressed independently into the chosen recipient parents employing the MABC approach. At ICAR-IARI, a set of 20 BC 3 F 3 lines were developed by crossing Pusa 372 with ICC 4958 followed by three backcrossing and two subsequent selfing generations ( Figure 1). Similarly, 20 BC 2 F 3 lines were developed by crossing Pusa 362 with ICC 4958, also at ICAR-IARI followed by two backcrossing and two subsequent selfing generations ( Figure 2). Similarly, 21 BC 3 F 3 lines were developed at ICAR-IIPR by crossing DCP 92-3 with ICC 4958 followed by three backcrossing and two subsequent selfing generations ( Figure 3).

Phenotyping of MABC introgression lines
A set of selected 20 BC 3 F 3 lines in the genetic background of Pusa 372 along with the donor and recipient parents were Another set of 20 BC 2 F 3 lines in the genetic background of Pusa 362 along with donor (ICC 4958) and recipient parents were phenotyped for root traits, phenological traits, and yield-related traits at the experimental farm of ICAR-IARI, New Delhi, during the year 2015-2016. Randomized complete block design with three replications under rainfed environment was conducted following all recommended cultural and agronomic practices. Each genotype was planted in a four -row plot of 5-m length with 10 and 30 cm between plants and rows, respectively. Root traits, that is, root dry weight (RDW [g]), root length (RL [cm]), root surface area (RSA [mm 2 ]), and specific root length (SRL [cm g −1 ]), were recorded as described earlier . Phenological traits and yield-related traits include PHT, DF, DM, PPP, biomass (g), harvest index (%), and YLD.
A total of 21 BC 3 F 3 lines along with recurrent (DCP 92-3) and donor (ICC 4958) parents were phenotyped under water-stress conditions at ICAR-IIPR, Kanpur, India (26˚26′59.7228′′ N, 80˚19′54.7356′′ E) during November to April 2015-2016. Random block design with plot size 4 × 0.3 m (1.2 m 2 ) in three replications and four blocks within replication were adopted for conducting the experiment. Approximately 40 seeds were maintained per row and recommended crop management practices were followed at the experimental location. Data were collected on plot basis on DF, DM, primary branch, secondary branch, tertiary branch, YLD, PPP, PHT, and 100SDW. The three best performing BC 3 F 3 lines-IPC(L4-14), IPC(L4-16), and IPC(L19-1)-were selected based on yield and other yield-attributing agronomic traits under moisture stress and further phenotyped under waterstress and non-stress conditions during November to April 2016-2017. The moisture stress was maintained depending on the rainfall throughout the cropping season and grain yield data was recorded for water stress and nonstress conditions and lines with lower drought susceptible index were identified.

Statistical analysis
Family-wise ANOVA was carried out using PROC GLM (SAS Institute, 2016) considering replication, MABC lines as fixed. Least-square means were calculated from analysis of variance.

Marker polymorphism, foreground, and background selection
Among eight SSR markers in the QTL-hotspot region tested for polymorphism, two markers (NCPGR21 and NCPGR127) were found polymorphic in the case of Pusa 372 × ICC 4958 and Pusa 362 × ICC 4958 combinations (Supplemental Table  S1). Hence these two markers (NCPGR21 and NCPGR127) were used for confirmation of hybrids in the F 1 generation and foreground selection in subsequent generations in the case of Pusa 362 × ICC 4958 (BC 1 F 1 , BC 2 F 1 ) and Pusa 372 × ICC 4958 (BC 1 F 1 , BC 2 F 1 and BC 3 F 1 ) crosses. In the case of DCP 92-3 × ICC 4958 combination, three polymorphic SSR markers (TAA170, NCPGR21, and TR11) were used for confirmation of hybrids in the F 1 generation and foreground selection in subsequent generations (BC 1 F 1 , BC 2 F 1 , and BC 3 F 1 ) (Supplemental Table S1).
For background selection, among the 346 SSR markers tested on the selected donor and recipient parents, 129 markers showed informative polymorphisms that can be used for background selection. Among the polymorphic markers, six to eight markers per linkage group that were equally distributed across the chickpea genome were selected and used for background selection. Thus, in total, 47, 53, and 46 polymorphic markers were used for background selection for Pusa 372 × ICC 4958, Pusa 362 × ICC 4958, and DCP 92-3 × ICC 4958 crosses, respectively (Supplemental Table S2).

3.2
Introgression of the QTL-hotspot genomic region in elite chickpea cultivars

Introgression of the QTL-hotspot genomic region in Pusa 372
A total of 18 F 1 seeds were harvested by crossing Pusa 372 × ICC 4958 in the postrainy season of 2013-2014 at the ICAR-IARI farm, New Delhi. These seeds were grown in the off-season nursery at the ICAR-IARI regional station, Dharwad in the rainy season of 2014-2015, and eight heterozygous F 1 plants identified using NCPGR127 and NCPGR21 markers were backcrossed with the recurrent parent and 12 BC 1 F 1 seeds were obtained. Of these 12 BC 1 F 1 seeds, based on foreground selection using NCPGR127 and NCPGR21, five BC 1 F 1 plants (with higher genome recovery using 47 SSR markers) were selected and then used for the second cycle of backcrossing in 2014-2015 at ICAR-IARI farm, New Delhi. Subsequently, approximately 98 BC 2 F 1 seeds were harvested. From the 98 BC 2 F 1 plants, 44 heterozygous plants selected using foreground markers were screened with 47 SSR markers for background selection (Supplemental Table S2). As a result, six BC 2 F 1 plants with high RPG recovery (90%) were used for a third backcrossing at ICAR-IARI regional station, Dharwad, to generate 108 BC 3 F 1 (Figure 1a). Upon background selection using 47 SSR markers, 20 BC 3 F 1 plants showing 96% of RPG recovery were selected for selfing. Finally, after two generations of selfing, 20 best BC 3 F 3 plants were evaluated based on foreground and background selection, as well as agronomic performance and evaluated at ICAR-IARI Delhi. The best-performing line, BGM 10216, was nominated for ICAR-AICRP on Chickpea trials.

Introgression of the QTL-hotspot genomic region in Pusa 362 variety
In the case of introgression of the QTL-hotspot genomic region in Pusa 362 variety, 13 F 1 seeds were harvested after crossing Pusa 362 with ICC 4958 at ICAR-IARI farm, New Delhi. Five true F 1 plants identified using NCPGR127 and NCPGR21 markers were backcrossed with the recurrent parent, and eight BC 1 F 1 seeds were obtained in a greenhouse during the off-season in 2012. Of these eight BC 1 F 1 , five BC 1 F 1 plants (with higher genome recovery using 53 SSR markers) were selected and then used for a second cycle of backcrossing during crop season 2012-2013. Subsequently, approximately 172 BC 2 F 1 seeds were harvested. Of the 172 BC 1 F 1 , 24 heterozygous plants selected based on foreground selection were screened with 53 SSR markers for background selection (Supplemental Table S2). As a result, four BC 2 F 1 plants with high RPG recovery (90%) were selfed to generate 134 BC 2 F 2 plants. Of these 134 BC 2 F 2 plants, 51 were found homozygous for both flanking markers (NCPGR127 and NCPGR21). Of 51 homozygous plants, 20 plants with 90-97% RPG were selected to raise BC 2 F 3 plants (Figure 2a). Based on agronomic performance, BG 3097 line was nominated for ICAR-AICRP on Chickpea trials.

3.2.3
Introgression of the QTL-hotspot genomic region in DCP 92-3 variety DCP 92-3 variety was targeted for introgressing the QTLhotspot genomic region at ICAR-IIPR, Kanpur. Of 34 F 1 plants derived from DCP 92-3 × ICC 4958 cross (crop season 2011-2012), 12 plants confirmed as true hybrids with foreground markers (TAA170, NCPGR21, and TR11) were used for making the first backcross, and 57 BC 1 F 1 seeds were harvested during crop season 2012-2013 (Figure 3a). Based on foreground selection, three BC 1 F 1 plants were selected and backcrossed with recurrent parent to generate 68 BC 2 F 1 seeds. Of the 68 BC 2 F 1 seeds harvested during 2013-2014, 18 BC 2 F 1 plants found positive for the QTL-hotspot alleles were selected for background selection. The RPG recovery varied between 65 and 70% among the 18 BC 2 F 1 plants using 46 markers. The selected 18 BC 2 F 1 plants were subjected to one more round of backcrossing and, subsequently, 182 BC 3 F 1 seeds were harvested during crop season 2014-2015. After foreground selection, 51 BC 3 F 1 plants were found positive and selfed to generate 168 BC 3 F 2 seeds. Of the 168 BC 3 F 2 plants, 75 BC 3 F 2 plants were analyzed with foreground markers and 21 BC 3 F 2 plants were found positive. The RPG recovery of 21 BC 3 F 2 plants ranged from 89 to 94% using 46 SSR markers (Supplemental Table S2).

Performance of Pusa 372 ILs
Twenty ILs (BC 3 F 3 lines) along with donor (ICC 4958) and recurrent parent (Pusa 372) were evaluated for different morphological, phenological, and yield-related traits under rainfed conditions during 2016-2017 at ICAR-IARI fields. Analysis of variance for the traits studied indicated a significant difference among the ILs (Supplemental Table S3). Although ILs differed significantly for DM, PPP, and YLD, they did not differ significantly from the recurrent parent for PHT and DF. Among 20 ILs, two ILs (Pusa 372_IL12 and Pusa 372_IL17) recorded maximum yield (22%) over the recurrent parent (Supplemental Table S4). The increased yield is a result of the increased number of PPP ( Figure 1b). Further, it was also noted that, all ILs recorded higher seed yield than both recurrent as well as donor parents. Among the ILs, 100SDW ranged from 20.19 (Pusa 372_IL05) to 24.32 g (Pusa 372_IL01) with an average of 22.7 g. Nevertheless, none of the ILs had 100SDW greater than the donor parent ( Figure 1c). The results indicate that, morphologically, the Under the ICAR-AICRP on Chickpea, a special trial known as 'drought tolerance introgression lines' (DTILs) evaluated the ILs developed by introgressing the QTL-hotspot into different genetic backgrounds. Among the best performing lines, one of the lines, Pusa 372_IL12 designated as BGM 10216, was evaluated in six and five locations during crop season 2017-2018 and 2018-2019 in AVT1 and AVT2, respectively. BGM 10216 outperformed Pusa 372 in four out of six locations in AVT1 and all five locations in AVT2 trials (Figure 1d). In overall performance, BGM 10216 recorded a highest mean yield 1,475 kg ha −1 with a potential 2,575 kg ha −1 under drought-stress conditions over the recurrent parent Pusa 372 with mean yield 1,272 kg ha −1 . It recorded an overall weighted percentage increase over the mean of 16% with 8% in AVT1 (Table 1) and 30% in AVT2 (Table 2). Advanced varietal trial data also indicated that it is an early flowering (50-55 d) and early maturing variety (106 d).

Performance of Pusa 362 ILs
Twenty BC 2 F 3 lines along with donor and recurrent parent (Pusa 362) were evaluated for root traits and phenological and yield-related traits. Except for the RDW trait, the ILs differed significantly for all other root traits under rainfed conditions (Supplemental Table S5). Some of the ILs had RL and RLD values greater than both donor and recurrent parents (Figure 2b and 2c) Table S8). The PPP in recurrent parent Pusa 362 was 48. Biomass per plot ranged from 415.6 (Pusa 362_IL07) to 800 g (Pusa 362_IL05) with mean of 561.58 g. Seed yield per plot ranged from 324 (Pusa 362_IL13) to 568.7 g (Pusa 362_IL05) with mean seed yield 418.8 g. The recurrent parent Pusa 362 yielded 521.33 g while the donor parent ICC 4958 gave a yield of 276 g. Pusa 362_IL05 was the best IL, which gave significantly higher yield than Pusa 362 under rainfed condition. Among the best performing lines, one of the lines, designated as BG 3097 (IL05), was evaluated in six locations during crop season 2017-2018 in AVT1 and at four locations during 2018-2019 in AVT2. BG 3097 recorded an overall weighted mean yield advantage of 8.0% over recurrent parent Pusa 362 in DTIL trial across 10 locations under AICRP over two consecutive years (AVT1 & AVT2; Table 3 and 4). Overall, BG 3097 showed mean yield superiority of 12.4% over national check JG 16 and 3.5% over JAKI 9218 under drought conditions. Overall, BG 3097 recorded a weighted mean yield of 1,374 kg ha −1 with a potential 1,902 kg ha −1 under drought-stress conditions over the recurrent parent Pusa 362, which recorded weighted mean yield of 1,272 kg ha −1 .

DISCUSSION
In the context of rapid climate changes, development of climate-resilient varieties is prerequisite to attain sustainable crop production and meet the global food demands. Achieving self-sufficiency and meeting future demands for food grains may not be possible using conventional breeding approaches alone. To accelerate faster genetic gains and make small-holder agriculture profitable, the integration of approaches like genomics, phenotyping, and systems modelling and agronomy are essential . Recently, the '5Gs' (genome assembly, germplasm characterization, gene function identification, genomic breeding, and gene editing) breeding approach has been proposed for achieving precision and enhancing the crop improvement to meet the future demands of nutritious food . Climate changes in recent years increased the occurrence of more severe, longer, and intense droughts in most of the areas in northern and eastern India (Ge, Huang, Xu, Qi, & Liu, 2014). Hence, breeding for drought-tolerant chickpeas is a priority. This a first study in pulses that reports enhancement of drought tolerance is multiple genetic backgrounds of chickpea as well as release of one drought-tolerant molecular breeding variety for commercial cultivation using the MABC approach. In the present study, we report development of 61 ILs (  Of eight SSR markers in the QTL-hotspot genomic region, none of the three donor and recipient parent combinations had all eight markers polymorphic. Hence, we used two to three polymorphic SSR markers with respective crosscombinations for foreground selection at each generation. The low percentage of marker polymorphism is not uncommon in highly self-pollinated species like chickpea. Three SSRs markers (TAA170, ICCM0249, and STMS11) were used for foreground selection while introgressing the QTL-hotspot genomic region into JG 11 genetic background. Further, based on marker polymorphism, we used 46 to 53 markers for estimating the RPG recovery. In the case of ILs in Pusa 362 background, 90-97% of RPG recovery was observed, hence the third cycle of backcrossing was not taken like the other two MABC programs. Earlier, higher background genome recovery in the second backcross generation was also reported in the case of molecular breeding lines with enhanced Fusarium wilt resistance developed in Annigeri 1 genetic background (Mannur et al., 2019). Similarly, higher genome recovery in early generations is not uncommon. For instance, in the case of rice (Oryza sativa L.), 91.6% RPG recovery in the BC 2 F 1 generation was reported while pyramiding blast resistance genes into an elite Basmati rice (Singh et al., 2013). In the case of ILs derived from Pusa 372 and DCP 92-3, >90% RPG recovery was observed. Similarly, >90% background genome recovery was also reported in earlier studies in chickpea in progenies after three backcross generations (Pratap et al., 2017;Varshney et al., 2013aVarshney et al., , 2014b. Pusa 372 is a popular and widely adapted landmark chickpea variety released for the northwestern plains zone, the northeaster plains zone, and the central zone in India. A total of 20 ILs were developed after three cycles of backcrossing of Pusa 372 with ICC 4958 followed by two selfings. Interestingly, all introgression lines outperformed both donor and recipient parents evaluated under rainfed conditions at ICAR-IARI. Large variation for DM was observed among the ILs. The early flowering and early maturing ILs identified in the present study will fit well in double cropping, therefore, these ILs are ideal for the sustainability of rice-based cropping systems. Further, because of short duration they also fit into cropping systems of the central zone. Pusa 362 is a bold-seeded and Fusarium wilt resistant variety released for cultivation in the northwestern plains zone in India by ICAR-IARI in 1995. A total of 20 ILs were generated after two cycles of backcrossing with Pusa 362 and two rounds of selfing. These ILs have shown significant variation in RDW and RLD. In an earlier study, 257 recombinant inbred lines derived from a cross between ICC 4958 (large root system) and 'Annigeri' (an agronomically elite variety) also showed variation for RLD at 35 d after sowing in field conditions under terminal drought (Serraj et al., 2004). Lynch and Wojciechowski (2015) have also considered RL and root depth as important root architectural traits that directly influence the acquisition of water and nutrients from the soil. Most of the ILs are expected to be phenotypically similar to each other and to the recurrent parent Pusa 362. However, significant variation among the ILs was observed for seed yield and PPP. Some studies have reported that yield potential is known to contribute to yield under water stress in several crops including chickpea (Pang et al., 2017;Pushpavalli et al., 2020;. The recurrent parent Pusa 362 yielded 521.33 g while the donor parent ICC 4958 gave a yield of 276 g. Two ILs (BG 3097 and BG 4005), which gave significantly higher yield than Pusa 362 under rainfed condition, were nominated for multilocation testing and future release under ICAR-AICRP on Chickpea.
DCP 92-3 is a lodging-and wilt-resistant variety with yellowish brown, medium bold seeds released by IIPR in 1997 for cultivation in Punjab, Haryana, Delhi, northern Rajasthan, and western Uttar Pradesh. A total 21 ILs (BC 3 F 3 line) were developed by MABC of DCP 92-3 with ICC 4958 after involving three cycles of backcrossing and two rounds of selfing. Based on 2 yr of yield evaluation, IPC(L4-14), IPC(L4-16), and IPC(L19-1) ILs with >16% yield increase over the recurrent parent were nominated for AICRP Chickpea trials. IPC(L4-14) with 11.0% yield over check and the recurrent parent in AVT1 trial has been promoted to AVT2 trial.
Besides developing superior lines with enhanced yield under rainfed or drought-stress condition through introgression of the QTL-hotspot into different genetic backgrounds, we also report the successful release of one improved variety, Pusa Chickpea 10216, in India (https://icar.org.in/ content/development-two-superior-chickpea-varietiesgenomics-assisted-breeding). Pusa Chickpea 10216 recorded an overall weighted mean yield advantage of 16% over recurrent parent Pusa 372 across all the centers tested in national DTIL trials under AICRIP over two consecutive years, 2017-2018 (8% over Pusa 372 over six locations) and crop season 2018-2019 (30% over Pusa 372 over five locations). It is a profusely branching variety with more pods per unit area with overall weighted mean yield of 1,475 kg ha −1 and has yield potential of 2,575 kg ha −1 under drought stress over the recurrent parent Pusa 372, which yielded 1,272 kg ha −1 . It is an early flowering (50-55 d) and early maturing variety (106 d) and fits in central and southern zones. Further, it is moderately resistant to Fusarium wilt, dry root rot, and pod borer.
In summary, the present study demonstrates that the introgression of the QTL-hotspot into three elite genetic backgrounds enhances drought tolerance and seed yield under drought stress conditions. Further, superior ILs developed in different genetic backgrounds can be tested for possible release as improved varieties. In addition to developing the superior lines, we also reported the release of improved varieties with enhanced drought tolerance and higher yield under drought stress.

AU T H O R C O N T R I B U T I O N S
C.B., S.T., K.R.S. supervised the crossing and selection of lines in breeding program. M.T. supervised the marker genotyping and data analysis for selection of lines. R.K.S., S.S. conducted the experiments. M.T., M.R., B.S.P., A.C., R.P., Y.T., B.M., P.S.S., A.K.S., S.K.C., G.P.D., N.P.S., R.K.V. contributed resources, data analysis and interpretation. A.R., A.V. did the statistical analysis. R.K.V. conceived the idea and provided the technical support and guidance to the overall research. All authors read and approved the MS.

C O N F L I C T O F I N T E R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.