Marè et al. Rice (2023) 16:2 Rice https://doi.org/10.1186/s12284-023-00619-2 RESEARCH Open Access Marker‑Assisted Introgression of the Salinity Tolerance Locus Saltol in Temperate Japonica Rice Caterina Marè1*, Elisa Zampieri2,3, Viviana Cavallaro4, Julien Frouin5,6, Cécile Grenier5,6, Brigitte Courtois5,6, Laurent Brottier5,6, Gianni Tacconi1, Franca Finocchiaro1, Xavier Serrat7, Salvador Nogués7, Mireia Bundó8, Blanca San Segundo8,9, Noemi Negrini4, Michele Pesenti4, Gian Attilio Sacchi4, Giacomo Gavina10, Riccardo Bovina10, Stefano Monaco2,11, Alessandro Tondelli1, Luigi Cattivelli1 and Giampiero Valè12* Abstract Background Rice is one of the most salt sensitive crops at seedling, early vegetative and reproductive stages. Varie- ties with salinity tolerance at seedling stage promote an efficient growth at early stages in salt affected soils, leading to healthy vegetative growth that protects crop yield. Saltol major QTL confers capacity to young rice plants growing under salt condition by maintaining a low ­Na+/K+ molar ratio in the shoots. Results Marker-assisted backcross (MABC) procedure was adopted to transfer Saltol locus conferring salt tolerance at seedling stage from donor indica IR64-Saltol to two temperate japonica varieties, Vialone Nano and Onice. Forward and background selections were accomplished using polymorphic KASP markers and a final evaluation of genetic background recovery of the selected lines was conducted using 15,580 SNP markers obtained from Genotyping by Sequencing. Three MABC generations followed by two selfing, allowed the identification of introgression lines achiev- ing a recovery of the recurrent parent (RP) genome up to 100% (based on KASP markers) or 98.97% (based on GBS). Lines with highest RP genome recovery (RPGR) were evaluated for agronomical-phenological traits in field under non- salinized conditions. VN1, VN4, O1 lines were selected considering the agronomic evaluations and the RPGR% results as the most interesting for commercial exploitation. A physiological characterization was conducted by evaluating salt tolerance under hydroponic conditions. The selected lines showed lower standard evaluation system (SES) scores: 62% of VN4, and 57% of O1 plants reaching SES 3 or SES 5 respectively, while only 40% of Vialone Nano and 25% of Onice plants recorded scores from 3 to 5, respectively. VN1, VN4 and O1 showed a reduced electrolyte leakage values, and limited negative effects on relative water content and shoot/root fresh weight ratio. Conclusion The Saltol locus was successfully transferred to two elite varieties by MABC in a time frame of three years. The application of background selection until ­BC3F3 allowed the selection of lines with a RPGR up to 98.97%. Physi- ological evaluations for the selected lines indicate an improved salinity tolerance at seedling stage. The results sup- ported the effectiveness of the Saltol locus in temperate japonica and of the MABC procedure for recovering of the RP favorable traits. Keywords Rice, Breeding, MABC, Salt tolerance, Saltol, Background selection, Recovery percentage, KASP markers *Correspondence: Caterina Marè caterina.mare@crea.gov.it Giampiero Valè giampiero.vale@uniupo.it Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Marè et al. Rice (2023) 16:2 Page 2 of 18 Introduction plants growing under salt condition by maintaining in Abiotic stresses negatively affect more than 50% of crop the shoots a low ­Na+/K+ molar ratio, which is essen- yields (Vij and Tyagi 2007) and major losses are caused tial for salt tolerance. The OsHKT1;5 gene located in by salinity (Chinnusamy et al. 2005). At least 33% of the Saltol region has been proposed to be the respon- arable lands are affected by salinization and more areas sible gene for the salinity tolerance provided by Sal- are expected to deteriorate in the coming years because tol (Kobayashi et al. 2017). OsHKT1;5 encodes for a of global climate changes (FAO 2016). Indeed, climate xylem-expressed ­Na+ selective transporter and acts by change is increasing sea water intrusion in coastal areas decreasing ­Na+ content in shoots and maintaining ­K+ and evaporation (www.​knowl​edgeb​ank.​irri.​org). Moreo- homeostasis (Ren et al. 2005). ver, excess of irrigation with incorrect soil drainage, use Salt-tolerant varieties at the seedling stage carry- of poor quality water containing high level of salts and ing the Saltol QTL include Nona Bokra, Pokkali and increased capillarity rise of saline groundwater contrib- Hasawi (Platten et al. 2013), together with Capsule, ute to exacerbate salinity-related problems (Daliakopou- a Bangladesh indica landrace (Rahman et al. 2016), los et al. 2016; Ismail et al. 2007). FL478, a recombinant inbred line derived from the Rice (Oryza sativa L.) is one of the most salt sensi- cross between the salt-tolerant variety Pokkali and the tive crops at seedling, early vegetative (Lutts et al. 1995) salt-sensitive IR29 (Walia et al. 2005), and IR64-Saltol and at reproductive stages (Ismail et al. 2007; Singh et al. (Ho et al. 2016). IR64-Saltol was developed at IRRI 2009). Salt causes two types of stress: osmotic stress with from a cross between IR64, a rice variety with high- consequent cell dehydration and ion stress affecting cell yield that has been widely cultivated around the world, ion homeostasis (Munns and Tester 2008). Both stresses and FL478, used as donor of Saltol (Ho et al. 2016). disturbing metabolic processes, affecting tiller number, Functional validation of Saltol QTL has been panicle length, spikelet number per panicle, negatively achieved using traditional marker assisted breeding influence plant growth, development and yield (Kakar programs for varietal improvement in indica subspe- et al. 2019; Khatun et al. 1995; Munns and Tester 2008; cies (Singh et al. 2018), as well as through transgenic Zeng and Shannon 2000). At every dS/m increase in soil technology by ectopically expressing Saltol QTL- electrical conductivity (EC) beyond the threshold value related salinity-induced genes in rice. These last valida- of 3.0 dS/m, the rice yield penalty is estimated approxi- tions include the intermediate filament (OsIF) encoding mately 12% (Grieve et al. 2012). Though salinity affects gene differentially expressed in genotypes with or with- rice plants at all growing and developmental stages, its out the Saltol QTL (Soda et al. 2018) and the Saltol effect on young seedlings should be considered as highly QTL-localized histone gene binding protein-1b (OsH- detrimental since it directly influences plant establish- BP1b) belonging to bZIP transcription factors (Das ment and eventually, yield. It is therefore relevant to et al. 2019). Rice plants modified to express these two develop varieties with salinity tolerance at the seedling genes exhibited improved tolerance towards multiple stage that would promote an efficient growth at the early abiotic stresses, thus reflecting the importance of the stages in salt affected soils, leading to healthy vegetative genes that make up the QTL. growth that can protect crop yield (Hoang et al. 2016). Several reports of Saltol QTL introgression into high Mechanisms of salinity tolerance in rice have been yielding indica rice varieties like IR64, BR11, BRRI widely studied (Horie et al. 2012), but the genetic archi- Dhan 28, as well as Basmati rice varieties, indicate tecture of this trait is complex (Frouin et al. 2018). improvement of salinity tolerance at the seedling stage Several genes involved in rice salt tolerance have been (Bimpong et al. 2016; Babu et al. 2017; Geetha et al. studied (Reddy et al. 2017; Chuamnakthong et al. 2019) 2017; Gregorio et al. 2013; Guo and Ye 2014; Hasan and numerous quantitative trait loci (QTLs) have been et al. 2015; Huyen et al. 2012, 2013; Krishnamurthy identified (Frouin et al. 2018; Negrao et al. 2011; Rah- et al. 2020; Nair and Shylaraj 2021; Yadav et al. 2020). man et al. 2016; Mirdar Mansuri et al. 2020). Consid- Conversely, to our knowledge only in a very recent ering salt tolerance at early developmental stages, a study (Bundó et al. 2022) the effects of the Saltol QTL major QTL named Saltol that accounts for 62–80% of introgression into japonica varieties have been evalu- phenotypic variation under salinity stress, has been ated. This study was designed to introgress the Saltol identified in an inbred line population (RIL) obtained QTL from donor parent IR64-Saltol into the temper- by crossing the salt tolerant indica landrace Pokkali ate japonica genetic background of two salt-susceptible and the salt sensitive indica cultivar IR29 (Bonilla et al. rice varieties, Vialone Nano and Onice, using Marker 2002; Thompson et al. 2010). The Saltol QTL is located Assisted Back-Cross (MABC) approach and thus fur- on chromosome 1 and confers capacity to young rice ther evaluate its effect on salt tolerance into temperate japonica rice genetic background. Marè et al. Rice (2023) 16:2 Page 3 of 18 Materials and Methods Marker‑Assisted Backcrossing Strategy Plant Materials To introgress the Saltol QTL into Onice and Vialone The used recurrent parents (RP) were Onice and Vialone Nano, a marker assisted back-cross (MABC) pro- Nano, two elite rice Italian varieties (Oryza sativa subsp. gram with foreground and background selections was japonica). Vialone Nano is a temperate japonica medium applied. In the initial cross, Onice and Vialone Nano grain variety grown in Italy since 1937; it was developed were employed as female, and IR64-Saltol as male. The by crossing the Vialone variety with the reduced-height hybridity of ­F1 plants was confirmed using the Saltol variety Nano. Onice is a temperate japonica long grain flanking SSRs markers, RM580 (IRGSP-1.0 position from variety, registered in 2011. IR64-Saltol (Oryza sativa bp 9,605,723 to 9,605,779) and RM493 (IRGSP-1.0 posi- subsp. indica) was used as donor parent for Saltol QTL. tion from bp 12,281,143 to 12,281,320) (Additional file 2: IR64-Saltol was developed at IRRI from a cross between Table S1). In the subsequent BC generations, until ­BC3F1, IR64, an elite indica line, and FL478, a breeding line car- the respective RP were used as the female parents (Fig. 1). rying the Saltol QTL with very high level of seedling The SSR markers used to check the ­F1 plants were also stage salt tolerance (Singh et al. 2018). FL478, derived used for the foreground selection of the ­BC1F1 plants. In from IR29 x Pokkali cross, contains < 1 Mb Saltol QTL the ­BC2F1 and ­BC3F1, the plant heterozygosity and the DNA segment from the salt tolerant parent Pokkali, presence of the right allele at the Saltol locus were evalu- located on chromosome 1 between 10.6 and 11.5 Mb ated by using two KASP (Kompetitive allele specific PCR) (Thompson et al. 2010). IR64-Saltol was derived from markers id_1007745 (position 10,690,930 bp on chromo- three rounds of marker assisted back-crosses targeting some 1) and K_id1008539 (position 12,591,394 bp on only the Saltol QTL on chromosome 1 (Ho et al. 2016). chromosome 1) flanking the Saltol locus. The same KASP Seeds of the IR64-Saltol donor parent utilized in the pre- markers were also used to select B ­ C3F2 plants homozy- sent work were kindly provided by IRRI. gous at the Saltol QTL. Fig. 1 Flowchart of the Marker Assisted Back Cross (MABC) scheme used for Saltol QTL introgression in temperate japonica rice varieties Onice and Vialone Nano. The selected ­BC3F4 were used for field evaluations and hydroponic assays Marè et al. Rice (2023) 16:2 Page 4 of 18 For the background selection, about 240 KASP mark- For SSR markers used for testing F ­1 and for fore- ers selected from the list of the 2000 validated rice ground selection of ­BC1F1, the PCR was performed in markers established by the Integrated Breeding plat- a 10 μl reaction mix containing 20 ng genomic DNA, form (https://​www.​integ​rated​breed​ing.​net) were tested 0.04 µM forward primer, 0.2 µM reverse primer, 0.1 µM on the 3 parents and a set of 96 polymorphic markers M13(FAM) primer 5′-CAC​GAC​GTT​GTA​AAA​CGA​C-3′, were identified (Additional file 3: Table S2). As detailed 0.4 mM dNTPs mix, PCR buffer (5×), 1.5 mM ­MgCl2, below, these markers were used to assess the propor- and 0.5 U of Taq DNA polymerase. The PCR was run for tion of the recurrent parent genome recovery (RPGR%). 20 cycles with touch-down comprising of denaturation Background selection was applied from the ­BC1F1 gen- for 45″ at 94 °C, followed by annealing for 45″ at 61/51 °C eration and the plants showing the highest RP% recov- decreasing by 0.5 °C every cycle, and primer elongation ery (Table 2), were backcrossed to Onice and Vialone for 45″ at 72 °C. This was followed by 24 cycles compris- Nano to produce the subsequent BC generations, up to ing of denaturation for 45″ at 94 °C, annealing for 45″ at ­BC3F1. The selected B ­ C3F1 plants were self-pollinated to 51 °C, and primer elongation for 45″ at 72 °C, sandwiched produce ­BC3F2 seeds. Each B ­ C3F2 plant was subjected to between an initial denaturation for five minutes at 94 °C foreground selection to identify homozygous plants for and the final extension for seven minutes at 72 °C. The both the foreground markers. The selected B ­ C3F2 plants amplified products were visualized by capillary electro- were subjected to background selection to assess those phoresis using the Genome Analyzer 3130. with the highest RP% recovery. The selected B ­ C3F2 plants For foreground selection of ­BC2F1, ­BC3F1 and ­BC3F2 were finally self-pollinated to obtain B ­ C3F3, which were generations, KASP-based genotyping was performed in a screened in background selection to identify the “Best 4 μl reaction consisting of 2 µl of genomic DNA [20 ng], Saltol introgression lines”. Selected B ­ C3F3 were self-polli- 2 µl of KASP 2X Master Mix and 0.055 µl of KASP Assay nated to produce B ­ C3F4 seeds that were finally genotyped supplied by LGC Genomics, and 0.032 µl M ­ gCl2 50 mM. with genotyping by sequencing (GBS) to precisely verify The analyses were carried out using the 7500 Applied the level of homozygosity with respect to the recurrent Biosystem instrument setting the allelic discrimination parental genome (Fig. 1). program in three steps: (1) pre-read step of 1′ at 60 °C; During the BCs, the MABC scheme was coupled to (2) amplification reaction consists of an hold of 15′ at an embryo rescue (ER) technique to fasten the process. 94 °C; 10 cycles of 20″ at 94 °C, 60″ from 61 to 55 °C in Briefly, 11 days after the cross the seeds under forma- the touch-down method decreasing by 0.6 °C, 26 cycles tion were harvested and deprived of glumes. Seeds were of 20″ at 94 °C and 1′ at 55 °C; (3) post-read for 1′ at surface-sterilized by washing with 70% v:v ethanol for 40 °C. From ­BC1F1 generation onward, a set of 96 KASP 1 min, followed by a washing with 30% v:v NaClO for markers, polymorphic between each recurrent parent vs 30 min and three rinses with sterile distilled water for IR64-Saltol, were run on chips on BioMark HD to assess 5 min. After this step, the embryos were harvested under the RP% recovery of each line. The chips were loaded fol- sterile conditions and put on embryo rescue media (MS lowing the standard protocol from Fluidigm. The PCR 0.5 ×) in jar for approximately 14 days at 26 °C for 14 h program had 3 steps: (1) 1800″ at 70 °C, 600″ at 25 °C and of light and 22.5 °C for 10 h in the dark. One liter media 900 s at 95 °C; (2) 1 cycle 10″ at 95 °C, 10″ at 57 °C and contained 2.2 g Murashige and Skoog medium, 20 g 10″ at 72 °C; 12 cycles 20″ at 94 °C and 60″ by touchdown sucrose, 250 mg MES (4-Morpholineethanesulfonic acid, from 65 to 57 °C with a cooldown final step at 20 °C. The 2-(N-Morpholino) ethanesulfonic acid hydrate) and 2.5 g fluorescence was read with Fluidigm SNP Genotyping gelrite. Then, the germinated plantlets were acclimatized Analysis software. by transferring them in pots filled up with peat moss (1 L), vermiculite (0.55 L), calcium carbonate (1 g/L) and Genotyping by Sequencing osmocote (3 g/L) and watered with tap water and kept at Genomic DNA was extracted from young leaves of intro- the same condition of temperature and light as above. gression lines and their respective parental lines by high- throughput automated methods using NucleoMag Plant Foreground and Background Selection kit (Macherey–Nagel) and its quality was verified on 1.5% All genomic DNA extractions were performed by sam- agarose gel. After Hoechst quantification, DNA concen- pling young leaves of 15 days old plants and following the trations were normalized. DNAs were digested individu- Mixed Alkyl Trimethyl Ammonium Bromide protocol ally with the ApeKI restriction enzyme. GBS sequencing (Risterucci et al. 2000). Ball bearings (3.0 mm) were used library was prepared by ligating the digested DNA to to crush frozen samples by shaking 30″ at a frequency of unique nucleotide adapters (barcodes) followed by PCR. 30 rpm in a MM300 Mixer Mill (Retsch, Germany). The Sequencing was performed using Illumina HiSeq3000. To DNA was quantified on agarose gel and Qbit fluorimeter. detect informative SNPs, fastq files were analyzed using Marè et al. Rice (2023) 16:2 Page 5 of 18 Tassel V5 pipeline and an alignment on the O. sativa or 14-days to salt stress (80 mM NaCl in the nutrient Nipponbare reference genome (MSU7) with Bowtie2. solution), while the other was kept in control conditions. Only polymorphic parental loci were kept and filtered on Aeration was applied to the solution both in the pre- and heterozygous rate (< 40%) and then imputed with Bea- salt-treatments. All hydroponic solutions were renewed gle v5.0. 15,580 imputed SNP were kept for analysis. The every three days to minimize nutrient depletion. Three percentage in base pair (%bp, Table 3) was calculated by tanks for each condition were used. attributing to each SNP a base number value. Each value Electrolyte leakage (EL) analyses were carried out was calculated by dividing by 2 the difference of position seven days after salt treatment as described by Campo between the SNP before and the SNP after. % bp is the et al. (2014). Briefly leaf blades from the two youngest sum of all donor’s SNP value by the total genome length. enlarged leaves of each plant were cut in 1 cm segments and washed in Milli-Q water. Segments were incubated Agronomic Evaluations in 15 mL tubes at 25 °C for 2 h in 10 mL of Milli-Q water BC3F4 seeds coming from seven Onice-derived lines in a shaker, thus, without removing the segments, elec- and from six Vialone Nano-derived lines were evaluated tro-conductivity of the solution (Ec1) was measured by together with the recurrent parents in field trials under a pH/mV meter (HI5221, HANNA instruments, Italy). non-salinized soil conditions to evaluate their main agro- After autoclaving the tubes containing the leaf segments nomic traits in reference agro-environments. The field for 20 min, Ec2 was measured and the EL was calculated experiments were set-up in two different sites, at the as (Ec1/Ec2) × 100. The effect of salt treatment was eval- CREA rice research farm in Vercelli (45° 19′ 18″ N; 8° 22′ uated calculating for each genotype the relative increase 06″ E) and at SIS rice farm in Malalbergo (Bologna) (44° of EL in the treated samples with respect to the control 41′ 50″ N; 11° 28′ 53″ E) during 2019 season. Field plots ones. Three independent experiments were performed of 9 ­m2 were organized in randomized blocks with three with three replicates from four plants each. and two replicates in Vercelli and Malalbergo, respec- For ­Na+/K+ molar ratio evaluation, plants were har- tively, and were managed according to common agricul- vested after seven days of salt treatment. To remove tural practices for rice cultivation in the area, with direct apoplastic ­Na+ and K ­ +, roots were washed twice for dry-seeding on May the 2nd and continuous flooding of 10 min at 4 °C in a 25 mM R ­ b2SO4 solution before dis- paddy from the three-leaf stage until the end of ripening secting the plant tissues (young leaves, old leaves, sheath stage (Monaco et al. 2021). Flowering date, plant height, and stem, and roots). Samples were oven-dried at 70 °C panicle length, seeds number per panicle, sterility and for 72 h, and the resulting weight (DW) was measured. paddy yield were evaluated with subsamples collection Tissue samples were digested in Teflon tubes filled with in each plot following the methods described in Volante 10 mL of 65% (v/v) ­HNO3 by a microwave digester sys- et al. (2017) for rice phenotyping. tem (MULTIWAVE-ECO, Anton Paar GmbH) by apply- ing two-steps power ramps (Step 1: to 500 W in 10 min, Hydroponic Assays of the Selected Lines and Parents maintained for 5 min—Step 2: to 1200 W in 10 min, Rice seeds were surface-sterilized (3 min in 70% v:v maintained for 15 min). After 20 min cooling, the min- EtOH, 30 min in 1.5% v:v NaClO and 0.02% Tween 20) eralized samples were transferred into polypropylene and, after five rinses with sterile distilled water in ster- test tubes and diluted (1:40) with Milli-Q bi-distilled ile conditions, were placed on filter paper saturated with water (Millipore). ­Na+ and ­K+ were measured by Induc- distilled water and incubated in the dark at 26 °C. Seven tively Coupled Plasma-Mass Spectrometry (ICP-MS; days later, seedlings were transferred into 10 L plastic Bruker AURORA M90 ICP-MS, Bruker Daltonik GmbH, tank (6 seedlings of each genotype in each tank) con- Bremen, Germany). An aliquot of a 2 mg L ­ −1 internal 72 89 159 taining a modified Yoshida solution (1.43 mM ­NH4NO3, standard solution ( Ge, Y, Tb) was added to both 0.51 mM ­K2SO4, 0.85 mM ­KH2PO4, 0.12 mM ­K2HPO4, samples and calibration standards to a final concentra- 0.75 mM ­CaCl2 ­2H2O, 1.64 mM M ­ gSO4 ­7H2O, 9.5 μM ­ −1. Possible polyatomic interferences were tion of 20 µg L ­MnCl2 ­4H2O, 0.075 μM (­NH4)6 ­Mo7O24 ­4H2O, 18.9 μM removed by Collision–Reaction–Interface with an H ­2 ­H3BO3, 0.15 μM ­ZnSO4 ­7H2O, 0.16 μM ­CuSO4 ­5H2O, flow of 70 mL ­min−1. Three independent experiments 35.75 μM ­FeSO4-EDTA, pH 5) and kept for a 7-day pre- were performed (three replicates from three plants each). treatment period in a growth chamber maintained at The stress injury score based on IRRI standard evalua- 26 °C and 80% relative humidity during the 16 h light tion system (SES; IRRI 2013) was recorded on the same period and at 22 °C, and 70% relative humidity during the plants at both seven and 14 days after salt treatment. 8 h dark period. Photosynthetic photon flux density was Shoot and root fresh weight (FW), dry weight (DW), as 400 μmol ­m−2 ­s−1. At the end of the pre-treatment, plants well as shoots and roots length were evaluated at the 14th were split into two groups and one was exposed for seven day. Leaf relative water content (RWC) was evaluated Marè et al. Rice (2023) 16:2 Page 6 of 18 according to Barrs and Weatherley (1962) at the end of heterozygous at markers RM580 and RM493 flanking the the 14-d treatment period. Briefly, blades from the two Saltol locus. After the background selection, 12 B ­ C1F1 youngest enlarged leaves from each plant were weighed lines were selected for the Onice backcross scheme, (FW), dipped in distilled water in a Petri dish and kept in with a RP% genome recovery between 46 and 72%, while the dark for 24 h at 26 °C. Turgid weight (TW) was meas- 10 ­BC1F1 lines were selected in case of Vialone Nano ured after gently patting dry the leaves with a paper towel. (RPGR% between 45 and 61%). These lines were used as Dry weight was measured after 48 h oven drying at 60 °C. males for the subsequent round of backcrosses. ­BC2 gen- RWC was calculated as [(FW-DW)/(TW-DW)] × 100. erated 374 B ­ C2F1 plants; foreground selection identified 131 plants heterozygous for markers id_1007745 and K_ Statistical Analysis id1008539 flanking the Saltol locus. After the background Field trials data were separately analyzed for each site and selection, 17 ­BC2F1 plants were selected for Onice back- recurrent parent through ANOVA and Dunnett’s post- crosses, with RPGR% of 75–90%, and 18 B ­ C2F1 plants hoc test using R environment (R Core Team 2013). for Vialone Nano backcrosses with RPGR% of 73–89%. Significant differences in RWC, S/R FW ratio, shoot These lines were used as males for the last backcross. ­BC3 and root length between plant grown in control or salt yielded 518 B ­ C3F1 plants; of these, 207 were identified as conditions were determined by Student’s t-test, evaluat- heterozygous for markers id_1007745 and K_id1008539. ing pairwise comparisons of mean differences. Data con- After the background selection, 29 plants were selected cerning EL and N ­ a+/K+ molar ratio were subjected to for selfing of which 14 from the Onice and 15 from the ANOVA analysis. The homogeneity of the variances was Vialone Nano backcrosses. Selected plants showed a checked with a Levene’s test. A Tukey post-hoc test was RPGR% from 89 to 98%. carried out with Bonferroni correction, and significance Selfing of ­BC3F1 produced a total of 968 ­BC3F2 plants; was set at p < 0.05. foreground selection identified 218 plants (119 for Onice and 99 for Vialone Nano) homozygous for mark- Results ers id_1007745 and K_id1008539. Then, the background Marker Assisted Introgression of Saltol into Onice selection carried out on these plants allowed the identi- and Vialone Nano fication of 21 lines for Onice with RP% recovery between Assessment of hybridity of ­ F1 plants and foreground 92.4 and 100%, and 19 lines for Vialone Nano with RP% selection of the ­BC1F1 plants was conducted using the recovery between 93 and 100% (Table 2). An additional Saltol flanking SSRs markers, RM580 and RM493 (Addi- round of background selection was carried out on 277 tional file 2: Table S1), while in the B ­ C2F1 and B ­ C3F1, the ­BC3F3 plants (133 Onice lines and 144 Vialone Nano evaluation of plant heterozygosity and foreground selec- lines). This final step led to the selection of 14 lines for tion were performed with the KASP markers id_1007745 Onice and 12 lines for Vialone Nano as the best ones for and K_id1008539 flanking the Saltol locus. percentage of recurrent parent genome recovery (Fig. 3, To perform the background selection, a panel of 96 Additional file 4: Table S3). These selected lines were sub- KASP markers polymorphic between recurrent (Onice jected to an additional selfing generation and foreground and Vialone Nano) and donor (IR64-Saltol) parents were selection to obtain ­BC3F4 lines (Table 2). selected. Among them, 92 markers were polymorphic between Onice and IR64-Saltol, while 86 were polymor- Genotyping by Sequencing (GBS) of Selected ­BC3F4 Lines phic between Vialone Nano and IR64-Saltol. The panel To achieve a better characterization of the genomic contained 27 markers localized on chromosome 1 and, landscape of the lines obtained by the MABC program, among them, 7 were located within the Saltol region selected lines were further subjected to GBS using 15,580 as defined by markers id1007745 and K_id1008539, bp SNP markers polymorphic between the Saltol donor 10,690,930 and bp 12,591,394 of the rice genome, respec- IR64-Saltol and the recurrent parents Onice and Vialone tively (Fig. 2; Additional file 3: Table S2). The remaining Nano (Additional file 5: Table S4). For the lines deriv- 69 markers were distributed on the other 11 chromo- ing from Onice, the GBS analysis was applied to the 14 somes, with a range of 4–9 markers per chromosome ­BC3F4 lines selected following the results of the forward (Table 1; Additional file 3: Table S2). and background selection. Similarly, for the lines derived To introgress the Saltol QTL, crosses between Onice from Vialone Nano, GBS was applied to the 12 B ­ C3F4 and Vialone Nano, both temperate japonica rice varie- lines. ties, and the indica variety IR64-Saltol were followed by GBS analysis highlighted a RP% genome recovery rang- backcrosses and selfing generations, as summarized in ing from 93.36 to 98.97% and the two best lines, O2 and Table 2. A total of 550 ­BC1F1 plants were obtained for VN1, showed a RPGR% of 98.94 and 98.97 for the Onice the two crosses; foreground selection yielded 117 plants and Vialone Nano genomes, respectively (Fig. 4, Table 3). Marè et al. Rice (2023) 16:2 Page 7 of 18 Fig. 2 Physical position on the Nipponbare genome (Os-Nipponbare-Reference-IRGSP-1.0) of the 27 KASP markers for chromosome 1 utilized for the background selection (left image). Position of the markers is reported with respect to the position of the Saltol QTL in the indicated mapping studies; markers used for the foreground selection are highlighted in red (right image) When recovery of the recurrent parent genomes obtained were maintained, to be then examined for salinity toler- from GBS data were compared with those obtained from ance at the seedling stage. Towards this purpose, the background selection made with the 96 KASP marker best ­BC3F4 lines identified by background selection and panel, an over-estimation of the RP% genome recovery GBS (7 Onice-derived lines and 6 Vialone Nano-derived was highlighted for the last procedure (Table 3). lines) were tested in field trials in non-stress conditions and compared to their respective recurrent parents In‑Field Evaluations of Lines (Table 4). For Onice lines, yield data showed signifi- These field trials were carried out under non-salinized cant differences in Vercelli site for 1 out of 7 lines with conditions with the purpose of identifying introgressed a lower value (7.37 t h­ a−1) compared with the recurrent −1 lines in which agronomical traits of the recurrent parents parent (10.57 t h­ a ). Moreover, statistically significant Marè et al. Rice (2023) 16:2 Page 8 of 18 Table 1 Distribution of the KASP markers used in the site. More precisely, 5 out of 7 new lines in Malalbergo background selection on the 12 rice chromosomes and 2 out of 7 lines in Vercelli showed higher values of Number of polymorphic KASP markers Chromosome plant height compared with the recurrent parent, while 2 lines in Vercelli showed a delay in the flowering date. 27 Os01 For Vialone Nano, significant differences were detected 7 Os02 for plant height in Malalbergo and for flowering date in 8 Os03 both sites, with 1 line out of 6 that anticipated flower- 4 Os04 ing in Malalbergo and 5 lines which delayed it in Vercelli. 5 Os05 After these agronomic evaluations in field conditions 9 Os06 three lines (O1, VN1 and VN4), were selected as the most 7 Os07 promising lines for commercial exploitation. These lines, 8 Os08 which had shown high RP% genome recovery (Table 3), 5 Os09 did not show significant differences from their respective 4 Os10 recurrent parent for any of the phenotypic traits consid- 7 Os11 ered in the field trials (Table 4). 5 Os12 Physiological Characterization of Introgression Lines A physiological characterization was carried-out on differences among some lines and Onice recurrent parent the three lines (O1, VN1 and VN4) selected after the were detected for plant height in both sites, and for pani- agronomical field evaluations in comparison to Onice cle length, seed number and flowering time in Vercelli and Vialone Nano recurrent parents, the respective Table 2 Number of plants analyzed and RPGR% in back-cross generations during the marker assisted introgression of Saltol QTL Generation Tested and selected plants Onice × IR64-Saltol Vialone nano × IR64- Saltol BC1F1 Tested plants 330 220 Heterozygous plants at flanking markers 78 39 Selected plants in background selection 12 10 RPGR (Os01 excluded) 46–72% 45–61% BC2F1 Tested plants 166 208 Heterozygous plants at flanking markers 57 74 Selected plants in background selection 17 18 RPGR (Os01 excluded) 75–90% 73–89% BC3F1 Tested plants 242 276 Heterozygous plants at flanking markers 63 144 Selected plants in background selection and selfed 14 15 RPGR (Os01 excluded) 89–98% 91–98% BC3F2 Tested plants 500 468 Homozygous plants at flanking markers 119 99 Selected plants in background selection and selfed 21 19 RPGR (Os01 excluded) 92–100% 93–100% BC3F3 Tested plants 133 144 Homozygous plants at flanking markers 62 48 Selected plants in background selection and selfed 14 12 RPGR (Os01 excluded) 93–100% 94–100% BC3F4 Tested plants 14 12 Homozygous plants at flanking markers 14 12 Selected and selfed plants 14 12 Tested plants indicate the number of analyzed plants, heterozygous/homozygous plants at flanking markers indicate the number of plants selected in foreground selection that have the marker alleles flanking Saltol locus in heterozygous or homozygous condition. RPGR% = 1 − (KASP heterozygous (not included in Saltol locus) /total KASP markers)* 100 Marè et al. Rice (2023) 16:2 Page 9 of 18 Fig. 3 Graphical representation based on the 96 KASP marker alleles of Onice and Vialone Nano “Best introgression B ­ C3F4 lines” (for Onice, from O1 to O14; for Vialone Nano, from VN1 to VN12) carrying HKT 1;5 Saltol QTL, depicting the extent of recovery of the carrier chromosome 1 Marè et al. Rice (2023) 16:2 Page 10 of 18 Table 3 Ranking of ­ BC3F4 lines for the RPGR% as indicated The symptoms of salt stress injuries after 14 days of salin- by the GBS results (% SNP donor) and comparison with ization on shoots of parental IR64-Saltol, Vialone Nano, performances of the 96 KASP markers panel Onice and VN1, VN4, O1 introgression lines are exem- ID_GBS % SNP donor* % SNP recurrent % bp from donor not plified in Additional file 1: Fig. S1. detected by KASP Salt conditions, already after seven days of exposure, markers provoked a marked increase in EL from the leaf cells of O2 1.06 98.94 1.04 all the genotypes (Fig. 6A). In the sensitive recurrent VN1 1.03 98.97 1.03 parents Vialone Nano and Onice, the effects were par- VN8 1.85 98.15 1.11 ticularly marked since EL raised by 178% and by 278%, O1 2.14 97.86 1.80 respectively as compared with the controls. In the three O5 2.57 97.43 1.50 introgression lines, the increase for EL values were simi- O6 2.57 97.43 1.49 lar to that of IR64-Saltol (+ 130%) and for VN1 and O1 O10 2.94 97.06 0.44 resulted significantly lower than those measured in the O11 3.01 96.99 0.50 sensitive parents. O8 3.21 96.79 0.70 Concerning RWC (Fig. 6B), while leaves of control O12 3.19 96.81 0.69 plants showed similar values in the range of 82–85%, a O9 3.17 96.83 0.67 significant decrease was recorded at 14 days in the plants VN4 3.14 96.86 0.00 exposed to salt. Under salinity, RWC reduction was lower O13 3.55 96.45 2.76 in IR64-Saltol (− 19%) and in the introgression lines VN4 VN10 3.74 96.26 1.39 (− 17%) and O1 (− 13%) as compared with to the sensi- O4 3.91 96.09 2.83 tive parents Vialone Nano (− 32%) and Onice (− 45%) VN12 3.78 96.22 0.56 that showed a RWC lower than 60%, indicative of a stress VN7 4.61 95.39 2.26 condition (Zhao et al. 2014). VN9 4.56 95.44 2.21 Considering the shoot/root FW ratio (S/R; Fig. 6C), VN11 4.16 95.84 0.48 only O1 line under salt stress showed a reduction (− 26%) VN2 4.16 95.84 1.35 which was significantly lower than that of Onice recur- VN3 4.16 95.84 1.36 rent parent (− 70%). The lowest reduction (− 11%) of S/R VN6 4.73 95.27 1.60 FW ratio was observed in IR64-Saltol donor parent. No VN5 4.73 95.27 1.59 significant effects of Saltol locus introgression were high- O3 5.12 94.88 0.84 lighted on shoot and root length (Fig. 6D). O7 6.34 93.66 2.06 In the blades of the young leaves of the three intro- gression lines and IR64-Saltol, ­Na+/K+ molar ratios O14 6.64 93.36 2.37 were significantly lower than those of Vialone Nano and Onice recurrent parents (Table 5). A similar picture was susceptible checks, and IR64-Saltol, the donor parent observed also in the blades of old leaves even if in this as well tolerant check. After seven days of salt stress tissue, the differences among the genotypes were less (80 mM NaCl), most of the genotypes, including the tol- evident. Considering the whole shoot, significant differ- erant donor parent IR64-Saltol, showed from 21 to 26% ences were observed only for VN1, while for the other of plants with SES score values of 7–9 (Fig. 5A). However, introgression lines, the ­Na+/K+ molar ratios were lower, in O1 line only less than 6% of the plants showed SES val- but not significantly, than that of the recurrent par- ues ≥ 7. After 14 days of exposure to salt, the symptoms ents, whereas high ­Na+/K+ molar ratios were observed measured by the SES score worsened in all the genotypes in sheaths and stems. Concerning roots, only O1 line (Fig. 5B). Nevertheless, for the VN4 and O1 lines about showed a N ­ a+/K+ molar ratio significantly lower than 62% and 57% of plants, respectively, were assigned to that of the recurrent parent. the tolerant (SES = 3) or moderately tolerant (SES = 5) group, while for their recurrent parents, Vialone Nano Discussion and Onice, only 40% and 25% of the plants, respectively, Rice is classified as the most salt sensitive cereal crop, showed SES scores ranging from 3 to 5. Conversely, the particularly at seedling and reproductive stages (Kakar SES score of VN1 did not differ significantly from Vialone et al. 2019; Zeng and Shannon 2000). Its cultivation is Nano recurrent parent. Regarding the SES parameter, it is particularly threatened by salt stress, which is currently interesting to highlight that, under the experimental con- worsened due to climate change that is causing rising of ditions here assayed, both VN4 and O1 resulted signifi- the sea level, thus bringing saline water to inlands and cantly more tolerant than Saltol donor line IR64-Saltol. exposing rice growing areas to saline conditions. Since Marè et al. Rice (2023) 16:2 Page 11 of 18 Fig. 4 Comparison of the performances of 96 KASP marker panel and GBS in evaluating the RP genome recovery in the selected B ­ C3F4 introgression lines. The blue bars represent the percentage value in bp of the donor rate based on the KASP markers used in the background selection, while the orange bars represent the percentage value in bp calculated on the GBS data but not detected by KASP markers several rice cultivation areas in Italy and Europe, among markers yielded lines harboring Saltol locus and high which those located near river deltas, are currently or RP% genome recovery in only three years with a mini- at risk of being exposed to salinity, a molecular breed- mal linkage drag. Several MABC-based works have used ing approach coupled with phenotypic evaluations under microsatellites in the foreground selection of the Saltol hydroponic conditions was adopted for the generation locus (Ganie et al. 2016; Krishnamurthy et al. 2020; Linh of salt tolerant introgression lines derived from crosses et al. 2012; Nejad et al. 2008; Yadav et al. 2020). We took between the salt tolerant indica IR64-Saltol line and two advantage from a high-throughput genotyping method salt sensitive japonica varieties. based on KASP markers, for both, foreground and back- The two japonica rice varieties used in this study, ground selections, performed during the MABC cycles. Vialone Nano and Onice, are of great economic impor- To maximize the recovery of recurrent background of the tance in Italy for local and export markets, but both are chromosome 1 carrying the Saltol QTL, 27 polymorphic susceptible to salt stress, e.g. Vialone Nano is reported KASP markers, including 7 markers within the 1.9 Mb as one of the most salt-sensitive rice Italian variety (For- QTL region, were used to assess the precise introgres- mentin et al. 2018). Introduction of salt tolerance into sion of the Saltol locus from the donor IR64-Saltol. The renowned rice varieties through conventional breeding MABC scheme adopted in the present work involved method frequently implies that the developed progenies three backcrosses, that theoretically would allow a recov- acquire unwanted traits due to linkage drag and pos- ery of the RP genome of 93.75%. After five rounds of sible negative effects on yield and grain quality traits of background selection, up to B ­ C3F3 generation, lines with rice (Ismail et al. 2007; Thompson et al. 2010). Marker- a final RPGR% ranging from 93 to 100%, as estimated assisted back-cross (MABC) is a simple and efficient of the basis of the KASP markers alleles were selected. methodology to introduce specific traits into otherwise Extension of the background selection also during the popular varieties, and has been already used to select two selfing generations uncovered lines that became salt tolerant rice varieties through the introgression of homozygous at additional loci for the RP alleles, thus Saltol locus both in the indica (Krishnamurthy et al. increasing the success in the recovery of the recurrent 2020; Yadav et al. 2020) and japonica (Bundó et al. 2022) parent genomes. Comparisons between the RPGR% esti- genetic backgrounds. mated using the roughly 90 polymorphic KASP markers In this work, the application of MABC coupled to fore- and 15,580 polymorphic SNPs obtained by GBS (Table 3), ground and background selection based on molecular highlighted a slight under-estimation of the residual Marè et al. Rice (2023) 16:2 Page 12 of 18 Table 4 The results and statistics of phenotypic evaluations of ­BC3F4 introgression lines and the recurrent parent Onice (A) and ­BC3F4 introgression lines and the recurrent parent Vialone Nano (B), tested in two experimental sites (Malalbergo and Vercelli). ANOVA and Dunnett’s test significant for p(F) < 0.05 Variable Unit Site Total avg ANOVA Dunnett’s test O1/all (a) lines*100 p(F) Recurrent parent O1 ­line LSD (index)(b) (A) Yield ­ a−1 Paddy 13% t h Malalbergo 6.21 0.075 6.46 6.76 1.18 109.6 Vercelli 10.1 0.022* 10.6 10.6 2.94 104.2 Plant height cm Malalbergo 84.1 < 0.01** 74.3 74.3 7.97 86.9 Vercelli 100.2 < 0.01** 94.5 92.7 10.7 91.8 Panicle lenght cm Malalbergo 13.8 0.146 13.5 13.2 3.57 95.0 Vercelli 16.6 0.030* 16 16.1 3.36 97.0 Seed number Number per panicle Malalbergo 71.2 0.766 72.2 69.0 20.0 97.2 Vercelli 92.8 < 0.01** 120.2 95.3 46.2 107 Flowering Days from 1st July Malalbergo 45.6 0.272 48.5 41.5 7.56 91.8 Vercelli 46.2 < 0.01** 43.0 47.3 4.55 101.3 Sterility Scale Malalbergo 1.19 0.707 1.0 1.0 1.43 82.6 Vercelli 1.13 0.410 1.0 1.0 1.09 87.7 Variable Unit Site Total avg ANOVA Dunnett’s test VN1/all VN4/all (c) (c) lines*100 lines*100 p(F) Recurrent VN1 ­line VN4 ­line LSD (index)(d) (index)(e) parent (B) Yield Paddy 13% t Malalbergo 6.26 0.09 6.42 6.08 6.35 1.82 97.1 101.4 ­ha−1 Vercelli 9.34 0.17 N.A.(f ) 8.51 9.33 − 93.3 102.3 Plant height cm Malalbergo 121 0.046* 120 113 114 23.8 93.2 94.1 Vercelli 132 0.21 132 136 129 13.6 102.9 97.6 Panicle lenght cm Malalbergo 19.7 0.29 21.2 19.5 19.8 3.21 100.5 102.1 Vercelli 19.2 0.53 18.8 20.1 19.2 2.99 104.1 99.5 Seed number Number per Malalbergo 120 0.87 128 110 117 51.2 92.4 98.3 panicle Vercelli 120 0.23 104 123 138 47.5 101.7 114.0 Flowering Days from 1st Malalbergo 48.5 0.018* 48.5 49.0 50.5 6.38 101.0 104.1 July Vercelli 52.7 < 0.01** 50.0 55.7 54.0 2.97 104.3 101.1 Sterility Scale Malalbergo 1.1 0.5 1.0 1.0 1.0 0.89 92.6 92.6 Vercelli 1.2 0.190 1.0 0.7 1.0 1.56 54.9 82.0 Asterisks indicate statistically significant differences (*p(F) < 0.05; **p(F) < 0.01) (a) O1 introgressed line selected for the hydroponic assessment (b) Average of O1 line/average of all Onice lines*100 (c) VN1 and VN4 introgressed lines selected for the hydroponic assessment (d) Average of VN1 line/average of all Vialone Nano lines*100 (e) Average of VN4 line/average of all Vialone Nano lines*100 (f ) N.A. not available donor genome (estimated as % of bp of the donor) by need to scan the whole genome and the cost of each sam- the KASP marker evaluations, ranging from 0.44% (line ple. Results obtained support that the regularly spaced O10) to 2.83% (line O4). GBS analysis was carried-out as 96 KASP markers employed in this work can effectively a-posteriori verification to assess whether the number of assess the recovery of the recurrent parent rice genomes polymorphic KASP markers that were used for the back- during the background selection. ground selection (about 90) was appropriate to run an Effectiveness of the background selection was also efficient marker-aided introgression. The 96 markers chip confirmed by the results of the phenotypic evalua- was considered a reasonable compromise between new tions in non-salinized field conditions, since most of the Marè et al. Rice (2023) 16:2 Page 13 of 18 Fig. 5 SES evaluation of the parental IR64-Saltol, the tolerant check, Vialone Nano (VN) and Onice (O), the susceptible checks and VN1, VN4, O1 introgression lines, after 7 days (A) and 14 days (B) of exposure to 80 mM NaCl. Data, expressed as the percentage of plants at each score value (1, highly tolerant; 3, tolerant; 5, moderately tolerant; 7, sensitive; 9, highly sensitive), are the means obtained from three independent experiments (12 plants/genotype each experiment) introgression lines did not differ significantly from their selected lines (O1, VN1, and VN4), compared to the respective recurrent parents for the six phenotypic traits respective recurrent parents showed, as a whole, lower considered in at least one of the two experimental grow- SES scores, reduced EL values, limited negative effects on ing sites chosen for the trials, with the exceptions of the both RWC and S/R fresh weight ratio, as well as a bet- plant height for some Onice lines and flowering time for ter ability to maintain relatively low N ­ a+/K+ molar ratio some Vialone Nano lines (Table 4). The field evaluation in the blade leaves indicating an improved salinity toler- and the RP% genome recovery results ended with the ance. Nevertheless, the different lines exhibited different selection of three lines, O1, VN1 and VN4, which did performances for the different parameters evaluated. The not show any significant difference with their respective O1 line performed as the best for SES, leaf RWC (even recurrent parent for the investigated traits, showed high better than IR64-Saltol) and shoot/root fresh weight RP% genome recovery and were the most promising for ratio, while for the VN lines, VN4 performed slightly bet- commercial exploitation. ter than VN1 for SES (at 14 days of salt treatment), for A physiological characterization was then conducted leaf RWC and for the shoot/root fresh weight ratio. Vari- using salt tolerance assays under hydroponic conditions. ability in performances among introgression lines was Early effects of salt stress on tissues include the loss of also observed for the N ­ a+/K+ molar ratios in the blades selective permeability of the plasma-membrane, leading of young and old leaves, where all the lines performed to a relative high leakage of electrolytes from the cell, as better than IR64-Saltol, while for the whole shoot, a posi- a symptom (Bajji et al. 2002; Demidchik et al. 2014). In tive effect was observed only for VN1. addition, the ability of maintaining relatively low N ­ a+/ This variability in the response to salinity could be + K molar ratios in the tissues photosynthetically more attributed to the complexity of salt tolerance character. active under stress condition, confers tolerance to the Indeed, the Saltol QTL comprises several genes poten- plants and it is suggested to be the main physiological tially contributing to salt tolerance including membrane trait associated to Saltol QTL (Bonilla et al. 2002; Gre- transporters, signal transducers, transcriptional fac- gorio 1997; Gregorio et al. 2002; Waziri et al. 2016). In tors (Bundó et al. 2022; Chen et al. 2021; Das et al. 2019; view of that, several physiological parameters, includ- Nutan et al. 2017, 2020). Additionally, it is also important ing low Electrolyte leakage (EL), the reduction in RWC, to underline that despite the Saltol QTL has been exten- higher values of shoot and root fresh weight and shoot sively employed for improvement of salinity tolerance in and root length (Ghosh et al. 2016), as well as low values rice breeding, the specific loci in Saltol responsible for of ­Na+/K+ molar ratios are used as indicators for relative salinity tolerance is still a matter of debate (Li et al. 2018; salt tolerance in plants. The results obtained for the three Walia et al. 2005). Currently, a role in conferring salinity Marè et al. Rice (2023) 16:2 Page 14 of 18 Fig. 6 Salt stress response parameters evaluated in tolerant parent IR64-Saltol, susceptible parents Vialone Nano (VN), Onice (O) and VN1, VN4, O1 introgression lines. Plantlets were grown for 7 days (A Electrolyte leakage evaluation) or 14 days (B Relative water content in leaves; C shoot/root fresh weight ratio; D shoot and root length) in modified Yoshida solution in the absence (blue columns, controls) or presence (orange columns) of 80 mM NaCl. Data are the means from three independent experiments (12 plants per genotype for each experiment); bars represent the SE values (n = 36). Student’s t-test (*p < 0.05). Values with the same letter(s) are not significant at p < 0.05, as resulted by a Tukey post-hoc test with a Bonferroni correction tolerance at the seedling stage has been demonstrated the 1.9 Mb QTL region (Fig. 3; Additional file 4: Table S3), only for OsHKT1;5 located in the Saltol QTL (Kobayashi supporting that intra-QTL recombination can occur et al. 2017) and to the transcription factor OsGATA8, also between Saltol flanking markers and the QTL region. localized within the Saltol QTL (Nutan et al. 2020). The introgression line VN1 showed the Vialone Nano Intra-QTL recombination could cause differential allele at the distal flanking marker id1007745, while still response of introgression lines carrying Saltol. In this maintaining the IR64-Saltol haplotype at markers flank- work, the 1.9 Mb QTL region was monitored with seven ing the OsHKT1;5 locus (Additional file 4: Table S3), as polymorphic markers, on average one marker every confirmed by the GBS data (Additional file 5: Table S4). 271 Kb, and recombination events were observed within However, the role of Saltol in salt tolerance was mainly Marè et al. Rice (2023) 16:2 Page 15 of 18 Table 5 Na+/K+ molar ratios in different tissues of VN1, VN4, O1 introgression lines and parental IR64-Saltol, the tolerant check, Vialone Nano (VN) and Onice (O), the susceptible checks Na+/K+ molar ratio Blade Sheath + stem Shoot Root Young leaves Old leaves IR64-Saltol 1.28 ± 0.15b 2.07 ± 0.18ab 7.46 ± 0.80a 3.19 ± 0.62a 5.32 ± 0.19a a a b a VN 1.81 ± 0.11 2.29 ± 0.05 4.06 ± 0.05 2.92 ± 0.13 1.46 ± 0.08c c c d b VN1 0.44 ± 0.11 1.29 ± 0.08 1.89 ± 0.14 1.68 ± 0.17 1.68 ± 0.06c bc c d ab VN4 0.87 ± 0.06 1.32 ± 0.02 2.40 ± 0.19 2.07 ± 0.21 1.07 ± 0.08c a a c a ONICE 2.25 ± 0.12 2.51 ± 0.13 3.35 ± 0.20 3.05 ± 0.24 2.73 ± 0.11b b b bc ab O1 1.00 ± 0.03 1.71 ± 0.08 3.60 ± 0.18 2.19 ± 0.22 1.29 ± 0.07c Data are the means ± ES of three independent experiments (three replicates for three plants each). For each trait, values with same letter(s) are not significantly different at p < 0.05, as resulted by a Tukey post-hoc test with a Bonferroni correction ascribed to the presence of OsHKT1;5, candidate gene donors in breeding programs to transfer the salt toler- responsible to confer the capacity in maintaining good ance phenotype to other japonica varieties. Finally, the ­Na+/K+ homoeostasis (Kobayashi et al. 2017; Ren et al. introgression lines can be also used in pyramiding pro- 2005;). Concerning this aspect, the effectiveness of the grams, where it will be possible to accumulate traits for introgression procedure adopted was confirmed, as all salt tolerance at seedling stage together with traits affect- the three selected lines proved more efficient than the ing salt tolerance at other phenological stages, thus com- recurrent parents, as well as than Saltol donor, in main- bining different salt tolerance QTLs in a single genetic taining a good ionic balance in the active photosynthetic background (Frouin et al. 2018). tissues (Table 5). The effectiveness of the Saltol region might also be Abbreviations affected by the genetic background of the recipient MABC Marker-assisted backcross parents. The background effect could be ascribable to GBS Genotyping by sequencing unknown interactions of Saltol locus with the genomic VN Vialone Nano O Onice regions of RP or to the existence of other minor QTLs KASP Kompetitive allele specific PCR and their interaction with Saltol to provide effective seed- SES Standard evaluation system ling stage salinity tolerance in the donor parent (Babu EL Electrolyte leakage RWC​ Relative water content et al. 2017; Singh et al. 2018). In several studies, indeed, EC Electrical conductivity no significant differences in salt tolerance responses SSR Single sequence repeat could be observed in Saltol-containing rice lines with FW Fresh weight DW Dry weight respect to lines without Saltol (Alam et al. 2011; De Leon TW Turgid weight et al. 2017; Han et al. 2020; Ho et al. 2016; Yadav et al. RP Recurrent parent 2020). ER Embryo rescue RPGR Recurrent parent genome recovery Conclusions Supplementary Information In this work the Saltol locus was successfully transferred The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12284-​023-​00619-2. to two elite Italian varieties by marker assisted backcross in a time frame of three years. The application of back- Additional file 1. Fig. S1. Visual symptoms of salt stress injuries (80 mM ground selection until B ­ C3F3 allowed the selection of NaCl) after 14 days of salinization on shoots of parental IR64-Saltol, Vialone lines with a RP genome recovery up to 98.97% based on Nano, and Onice and the introgression VN1, VN4, O1 lines. Results from a representative experiment are shown. 15,580 polymorphic SNP markers. Physiological evalua- tions for the three selected introgression lines obtained Additional file 2. Table S1. Saltol-linked SSR markers, used for foreground selection on ­F1 and ­BC1F1 generations and their physical position on chro- from the MABC procedure indicate an improved salin- mosome 1; primer nucleotide sequences, and physical locations within ity tolerance at the seedling stage. The best introgres- the Saltol QTL are provided. sion lines are currently under the path for their release Additional file 3. Table S2. List of the 96 polymorphic KASP markers used for commercial production; in addition to this exploit- for the background selection. Chromosomal position (in bp), alleles of the ing avenue, they can be used as elite japonica germplasm Marè et al. Rice (2023) 16:2 Page 16 of 18 Received: 6 April 2022 Accepted: 5 January 2023 KASP markers in the recurrent parents (Onice and Vialone Nano) and in the Saltol donor line (IR64-Saltol) are shown. Additional file 4. Table S3. Diagrammatic representation of the KASP marker alleles in the selected Onice-derived (from O1 to O14) and Vialone Nano-derived (from VN1 to VN12) ­BC3F4 introgression lines. References Alam R, Sazzadur Rahman M, Seraj ZI, Thomson MJ, Ismail AM, Tumimbang- Additional file 5. Table S4. Diagrammatic representation of the SNP Raiz E, Gregorio G (2011) Investigation of seedling-stage salinity tolerance marker alleles derived from GBS in the selected Onice-derived (from O1 QTLs using backcross lines derived from Oryza sativa L. Pokkali. Plant to O14) and Vialone Nano derived (from VN1 to VN12) B ­ C3F4 introgression Breed 130:430–437. https://​doi.​org/​10.​1111/j.​1439-​0523.​2010.​01837.x lines. Babu NN, Krishnan SG, Vinod KK, Krishnamurthy SL, Singh VK, Singh MP, Singh R, Ellur RK, Rai V, Bollinedi H, Bhowmick PK, Yadav AK, Nagarajan M, Singh Acknowledgements NK, Prabhu KV, Singh AK (2017) Marker aided incorporation of Saltol, a The technical assistance of Dr. Marco Canella (Council for Agricultural Research major QTL associated with seedling stage salt tolerance, into Oryza sativa and Economics-Research Centre for Cereal and Industrial Crops, s.s. 11 to “Pusa Basmati 1121.” Front Plant Sci 8:41. https://​doi.​org/​10.​3389/​fpls.​ Torino, km 2.5, 13100 Vercelli, Italy) is fully acknowledged as well as graphic 2017.​00041 support provided by Renzo Alberici. Bajji M, Kinet JM, Lutts S (2002) The use of the electrolyte leakage method for assessing cell membrane stability as a water stress tolerance test in Author Contributions durum wheat. Plant Growth Regul 36:61–70. https://​doi.​org/​10.​1023/A:​ CM, BC, GAS, GV conceived and supervised the work. MC, EZ, VC, GT, FF, XS, SN 10147​32714​549 developed the genetic materials. CM, EZ, JF, CG, BC, LB, AT performed and sup- Barrs HD, Weatherley PE (1962) A re-examination of the relative turgidity tech- ported the molecular analyses. RB, GV, SM performed the field evaluations. MB, niques for estimating water deficits in leaves. Aust J Biol Sci 15:413–428. BSS, NN, MP, GAS performed and supported the physiological evaluations. CM, https://​doi.​org/​10.​1071/​BI962​0413 EZ, BC, BSS, GAS, SM, AT, LC, GV contributed in writing and critically revised the Bimpong IK, Manneh B, Sock M, Diaw F, Kofi N, Amoah A, Ismail AM, Gregorio manuscript. All the authors read and approved the final manuscript. G, Singh RK, Wopereis M (2016) Improving salt tolerance of lowland rice cultivar ‘Rassi’ throughmarker-aided backcross breeding in West Africa. Funding Plant Sci 242(2016):288–299. https://​doi.​org/​10.​1016/j.​plant​sci.​2015.​09.​ We acknowledge the support of NEURICE project (New commercial European 020 RICE (Oryza sativa) harbouring salt tolerance alleles to protect the rice sector Bonilla P, Dvorak J, Mackill DJ, Deal K, Gregorio G (2002) RFLP and SSLP map- against climate change and apple snail (Pomacea insularum) invasion, Grant ping of salinity tolerance genes in chromosome 1 of rice (Oryza sativa L.) Agreement n. 678168). using recombinant inbred lines. Philipp Agric Sci 85:68–76 Bundó M, Martín-Cardoso H, Pesenti M, Gómez-Ariza J, Castillo L, Frouin J, Ser- Availability of Data and Materials rat X, Nogués S, Courtois B, Grenier C, Sacchi GA, San Segundo B (2022) All data generated or analysed during this study are included in this published Integrative approach for precise genotyping and transcriptomics of salt article [and its supplementary information files]. tolerant introgression rice lines. Front Plant Sci 12:797141. https://​doi.​org/​ 10.​3389/​fpls.​2021.​797141 Campo S, Baldrich P, Messeguer J, Lalanne E, Coca M, San Segundo B (2014) Declarations Overexpression of a calcium-dependent protein kinase confers salt and drought tolerance in rice by preventing membrane lipid peroxidation. Ethics Approval and Consent to Participate Plant Physiol 165:688–704. https://​doi.​org/​10.​1104/​pp.​113.​230268 Not applicable. Chen T, Shabala S, Niu Y, Chen Z-H, Shabala L, Meinke H, Venkataraman G, Pareek A, Xu J, Zhou M (2021) Molecular mechanisms of salinity tolerance Consent for Publication in rice. Crop J 9:506–520. https://​doi.​org/​10.​1016/j.​cj.​2021.​03.​005 Not applicable. Chinnusamy V, Jagendorf A, Zhu JK (2005) Understanding and improving salt tolerance in plants. Crop Sci 45:437–448. https://​doi.​org/​10.​2135/​crops​ Competing interests ci2005.​0437 The authors declare no competing interests. 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