Map-based cloning of a salt tolerance gene Ncl and its utilization for improvement of salt tolerance in soybean

Description

Soybean [Glycine max (L.) Merr.] is the world’s primary crop source for protein and oil. Its cultivation is conducted in a wide range of environments and is exposed to many biotic and abiotic stresses that influence the sustainability of soybean production. Salt stress inhibits soybean growth and reduces grain yield. Genetic improvement of salt tolerance is essential for sustainable soybean production in saline areas. In this study, we isolated a quantitative trait locus (QTL) for salt tolerance using the map-based cloning strategy from a Brazilian soybean cultivar FT-Abyara to facilitate its use in soybean breeding.

To identify the gene that conditioned the salt tolerance QTL, we conducted fine mapping using 5,828 plants derived from F₉ residual heterozygous plants, and delimited the QTL to a 16.6-kb interval between markers SSR25.8 and CAPS42.4. Since only one predicted gene, Glyma03g32900, existed within the 16.6-kb region (Fig. 1), Glyma03g32900 was thus determined as the causal gene underlying the salt tolerance QTL (Ncl locus). The salt-tolerant soybean lines showed higher expression of the salt tolerance gene Ncl in the root and resulted in lower accumulations of Na⁺, K⁺, and Cl⁻ in the shoot under salt stress. We transformed Ncl full-length cDNA driven with 35S promoter (35S:Ncl) into a Japanese soybean cultivar Kariyutaka using the Agrobacterium-mediated transformation method, and significantly enhanced its salt tolerance (Fig. 2). To determine the usefulness of Ncl in conventional soybean breeding, we conducted introgression of Ncl into a salt-sensitive variety Jackson through continuous backcross, followed by DNA marker-assisted selection (MAS) using primers Satt339, SSR222042, and SSR112166 in each generation, and finally produced an improved salt tolerance line (Fig. 3). This result demonstrated that the DNA markers around Ncl could be used for introgression of Ncl into a salt-sensitive cultivar for developing a soybean variety with high salt tolerance. To determine the effect of Ncl in salt stress field conditions, we evaluated the Ncl near isogenic lines (NILs) in a salt stress field condition. The field experiments, which were conducted over three years, showed that Ncl could increase soybean grain yield by 3.6–5.5 times in saline field conditions (Fig. 4).

Our study suggests that using Ncl in soybean breeding through transgenic or MAS would contribute to sustainable soybean production in saline-prone areas.

Figure, table

Fig. 1. Fine mapping (n = 5,828) delimits Ncl to a 16.6-kb region between markers SSR25.8 and CAPS42.4 on chromosome 3.
Fig. 2. Overexpression of Ncl in transgenic soybean lines enhanced its salt tolerance.
Fig. 3. Introgression of Ncl into a salt-sensitive variety “Jackson” by marker-assisted selection (MAS) produced an improved salt tolerance line.
Fig. 4. Field performances of Ncl near isogenic lines (NILs) in a saline field condition in Miyagi Prefecture, Japan.
(a) Top view of N18-61 and N18-99 grown in a salt stress field in 2012. (b) Grain yield results of the NILs in a salt stress field in 2012. N18-39, N18-99, and N18-122 are lines carrying the tolerant allele of Ncl, whereas N18-9, N18-61, N18-180 are lines that had the sensitive allele. Data are shown as mean ± s.d. from three replicates. **: Significant difference (P < 0.01) based on ANOVA (Tukey’s multiple comparison test).