Marker-Assisted Introgression of the Salinity Tolerance Locus Saltol in Temperate Japonica Rice

Abstract

Background: Rice is one of the most salt sensitive crops at seedling, early vegetative and reproductive stages. Varieties 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 achieving 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%. Physiological evaluations for the selected lines indicate an improved salinity tolerance at seedling stage. The results supported the effectiveness of the Saltol locus in temperate japonica and of the MABC procedure for recovering of the RP favorable traits.

Keywords: Background selection; Breeding; KASP markers; MABC; Recovery percentage; Rice; Salt tolerance; Saltol.

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

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)

Fig. 3

Graphical representation based on the 96 KASP marker alleles of Onice and Vialone Nano “Best introgression BC₃F₄ 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

Fig. 4

Comparison of the performances of 96 KASP marker panel and GBS in evaluating the RP genome recovery in the selected BC₃F₄ 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

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)

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