(c) Chromosomal constitutions of five recombinants (lines 1624, 2171, 2555, 3571 and 8927) are shown with their salt tolerance. Positional cloning narrowed the GmSALT3 locus to a 17.5‐kb region between QS100001 and QS1119, and only one gene is predicted to be located in this region. Red and green represent homozygous Tiefeng 8 and 85–140, respectively, grey represents heterozygous.

We constructed a population of 367 recombinant inbred lines (RILs) derived from the F 2 population of a cross between the salt‐tolerant variety Tiefeng 8 and the salt‐sensitive variety 85–140 (Figure 1 a). The dominant gene associated with the salt‐tolerance phenotype was mapped between indel markers QS1101 and QS100011 on chromosome 3 (Figure 1 b), and was named GmSALT3 . To fine map the GmSALT3 locus, we self‐pollinated the F 5 plants heterozygous between indel markers QS1101 and QS100011, and planted the F 5:6 population (5769 individuals) in the winter of 2010 on Hainan Island, China. Seventy‐four recombinants between QS1101 and QS100011 were identified, and we determined their salt tolerance phenotypes in 2011 and 2012 by progeny testing. We obtained five plants containing recombinants between two markers QS100001 and QS1119. Two recombinants exhibited salt sensitivity and two exhibited salt tolerance, while line 3751 was segregating for the phenotype. The progeny testing of the recombinant plants allowed the locus GmSALT3 to be mapped to a 17.5‐kb region between indel marker QS100001 and cleaved amplified polymorphic sequence marker QS1119 (Figure 1 c, Table S1 in Supporting Information). There was only one gene predicted to be present within this 17.5‐kb region according to the soybean reference genome that has been obtained from the variety Williams 82. This gene, Glyma03g32900.1 , was regarded as the candidate causal gene underlying GmSALT3 . To explore whether variation in the salt‐tolerance phenotype was due to a difference in the coding sequence of Glyma03g32900.1 , RNA sequencing (RNA‐seq) of two RNA pools consisting of either 20 salt‐sensitive or 20 salt‐tolerant F 6 plants derived from the cross between 85–140 and Tiefeng 8 was performed. The results indicated that the GmSALT3 cDNA obtained from Tiefeng 8 was 2640 bp in length, consisting of an open reading frame (ORF) of 2436 bp with 5′ and 3′ untranslated regions (UTR) of 50 and 204 bp, respectively, whilst the ORF from 85 to 140 was 1131 bp (Figure S1a). After comparing the genomic sequence in the two parents it was apparent that there was a 3.78‐kb fragment inserted in exon 3 of Gmsalt3 in 85–140, comprising of long terminal repeats (LTRs) of length 643 and 647 bp that had 99.1% similarity to each other. The element was flanked with a 5‐bp target‐site duplication sequence (CATGG) and reverted 2‐bp repeat (TG … CA) (Figure 1 d). This resulted in a truncated Gmsalt3 transcript in 85–140 yielding only 376 amino acids (Figure S1b).

It is noteworthy that the expression pattern of GmSALT3 was different from that of AtCHX20 , the closest functionally characterized homolog of GmSALT3 , expressed in stomatal guard cells (Padmanaban et al ., 2007 ). However, as was found for AtCHX20 (Chanroj et al ., 2011 ), we observed an endomembrane (ER) localization for the GmSALT3 protein. This was initially determined by transiently expressing P35S:GmSALT3‐GFP (green fluorescent protein) in Nicotiana benthamiana leaves with more than 90% of the protoplasts expressing the construct displaying clear tubular and sheet‐like structures (Figure 4 a). This contrasts with the expression of free GFP where more than 90% of the protoplasts displayed cytosolic and nuclear fluorescence (Figure 4 d). To further assess the localization, GmSALT3‐GFP was co‐expressed with mCherry with an ER retention sequence (Figure 4 g). As shown in Figure 4 (i), GFP signals overlapped with the mCherry signals, consistent with GmSALT3‐GFP being localized to the ER, whereas cytosolic GFP did not overlap with ER‐localized mCherry (Figure 4 f). Furthermore, N‐ and C‐terminal YFP (yellow fluorescent protein) fusions of GmSALT3 were transiently expressed in Arabidopsis mesophyll protoplasts and were found to co‐localize with a fluorescent ER marker not a late endosomal/vacuolar marker (Figure S5).

(b) In situ PCR in sections (60 μm) of roots of a 4‐week‐old Tiefeng 8 soybean plant grown in 50/50 perlite/vermiculite with no salt treatment. Blue‐stained cells are where transcripts are present. Negative controls (b) without RT (reverse transcription) were included to show lack of genomic DNA contamination.

(a) Expression of GmSALT3 analysed using quantitative real‐time PCR (qRT‐PCR) in root, hypocotyl, stem, leaf and cotyledon tissue of Tiefeng 8 and 85–140. The numbers (0, 6 h; 1, 3, 5 days) indicate time points after growing plants under control or salt stress (200 mm NaCl). Transcription levels was calculated as a percentage of the GmUKN1 transcript. Error bars indicate standard deviations ( n = 3). Different letters indicate significant differences between treatments for a given organ according to Dunan's multiple range test at P < 0.05.

We investigated the expression pattern of GmSALT3 by quantifying the relative abundance of the mRNA in different organs of Tiefeng 8 and 85–140. The expression was much higher in roots than shoots of Tiefeng 8, while the transcripts were not detectable in tissues of 85–140 (Figure 3 a). After 6 h of 200 mm salt treatment the transcript abundance of GmSALT3 decreased in Tiefeng 8; however, after 3 days it recovered to higher levels (Figure 3 a). Using in situ PCR, as shown in Figure 3 , we detected the expression of GmSALT3 predominantly within endodermal cells and cells associated with phloem and xylem of salt‐tolerant Tiefeng 8 soybean root (Figure 3 b,c) and within proto‐phloem cells in young secondary root (Figure 3 d); the localization was unchanged under salinity treatment (Figure S2). In stems and leaves, GmSALT3 shared a similar expression pattern as in the root (Figure S3). In order to corroborate cell localization we cloned the putative promoter of GmSALT3 and expressed GmSALT3pro:: β ‐glucuronidase in A. thaliana ; GUS was detected mainly in vascular tissues of root, hypocotyls and leaves (Figure S4).

(a) Phylogenetic tree based on multiprotein sequence alignment of GmSALT3 to Arabidopsis CHX proteins and other related proteins. Multiprotein sequence alignment (Geneious Alignment) was achieved using Geneious Pro version 5 (Drummond et al ., 2011 ). All the protein sequences were obtained from the NCBI database ( http://www.ncbi.nlm.nih.gov/protein/ ). At, Arabidopsis thaliana ; Sc, Saccharomyces cerevisiae ; Vv, Vitis vinifera ; Cs, Cucumis sativus ; Gm, Glycine max ; Sl, Solanum lycopersicum ; Mn, Morus notabilis ; Mt, Medicago truncatula . GmCHX20* indicates CHX20 from the sequenced soybean cultivar Williams 82. The scale bar denotes the scale of amino acid substitutions.

Basic local alignment search tool (blastx, NCBI, http://blast.ncbi.nlm.nih.gov ) (translated) analysis of the GmSALT3 cDNA sequence showed that GmSALT3 shared 73% identity with an uncharacterized protein annotated as a K + /H + antiporter (MTR_7g099820) from Medicago truncatula and 59% identity with the characterized Arabidopsis thaliana AtCHX20 (Figure 2 a). Accordingly, GmSALT3 had a confidently predicted ‘sodium/proton exchanger’ (NHE) domain (Pfam00999, e ‐value of 2.9e‐69), a diagnostic of cation/proton exchangers in plants (Chanroj et al ., 2011 ), that started near the N‐terminus between amino acids 30 and 428 as well as 10 predicted transmembrane domains (TMDs) (Figures 2 b,c and S1b). The truncation of GmSALT3 (at amino acid 370) lies between TMD 9 and TMD 10, which would result in the loss of the TMD 10 domain and the C‐terminus.

(b) The Na + content in the stem and leaf of non‐grafted, self‐grafted and reciprocally grafted lines NIL‐T ( GmSALT3 ) and NIL‐S ( Gmsalt3 ) under salt stress for 8 days. TCK and SCK: NIL‐T and NIL‐S, respectively, under control conditions. Data are means of three replicates ± SE.

(a) The Na + content in Tiefeng 8 (blue) and 85–140 (red) under control or NaCl stress (200 mm NaCl) for 0, 1, 3, 5 or 7 days. Data are means of three replicates ± SE. Asterisks indicates a significant difference between Tiefeng 8 and 85–140 at * P < 0.05, ** P < 0.01.

Salt tolerance in soybean is associated with limiting Na + accumulation in shoots (Liu et al ., 2011 ; Jiang et al ., 2013 ). To investigate the role of GmSALT3 , the Na + accumulation within the two parents was compared. The Na + content in roots of the parents was similar, but following NaCl treatment (200 mm), Tiefeng 8 accumulated significantly less Na + than 85–140 in both stems (after 5 days) and leaves (after 7 days) (Figure 5 a). To compare the function of the two GmSALT3 / Gmsalt3 alleles, we developed a pair of near isogenic lines (NILs) NIL‐T ( GmSALT3 ) and NIL‐S ( Gmsalt3 ). Both NIL‐T and NIL‐S were derived from a single F 6 plant of a cross between 85–140 and Tiefeng 8 that was heterozygous for the GmSALT3 locus. These lines had no polymorphism among 147 simple sequence repeat (SSR) markers distributed throughout the genome except for those located within the GmSALT3 locus. Under control conditions, the NILs had no significant difference in agronomic traits, such as 100‐seed weight, protein and oil content, but had differential salt tolerance (Figure S6). The Na + content in stems and leaves of self‐grafted NIL‐S was much higher than that in NIL‐T (Figure 5 b). When the NIL‐S scion was grafted on the NIL‐T rootstock, the Na + content in stems and leaves decreased by 48.7 and 70.65%, respectively, compared with the self‐grafted NIL‐S. In contrast, the Na + content in stems and leaves of the NIL‐T scion grafted to the NIL‐S root increased by 79.0 and 139.1%, respectively, compared with self‐grafted NIL‐T (Figure 5 b). These results suggest that GmSALT3 is likely to function in the root (and hypocotyl) and constrain Na + translocation to the lamina; this is consistent with the predominant expression pattern of GmSALT3 in Tiefeng 8 in roots and hypocotyls (Figure 3 a).

Geographical distribution of haplotypes reveals that the salt‐tolerant H1 is a likely target of natural and artificial selection

To identify allelic variation, the coding region of the GmSALT3 locus from 31 soybean landraces was sequenced. Five haplotypes (H) were observed in these accessions including the haplotypes found in Tiefeng 8 (H1) and 85–140 (H2) (Table S2). Of the newly identified haplotypes, when compared with H1, H3 had nine non‐synonymous SNPs, H4 had seven non‐synonymous SNPs and an 18‐bp deletion in exon 3 due to a nucleotide substitution (AG to AT) that was 3′ of the intron 2 splicing site and H5 had a 4‐bp deletion in exon 2 that resulted in a premature stop codon (Figure 6a). The promoter region starting 540‐bp upstream of the start codon was also sequenced; this which identified eight SNPs and three indels of 1, 4 and either 148 or 150 bp. For the fixed variation between the salt‐tolerant and salt‐sensitive haplotypes, two insertions of 148 and 4 bp were observed in the promoter region 152 and 103 bp before the start codon, respectively, in H3 and H4, whilst insertions of 150 and 4 bp were identified at those same locations in the promoter region of H5 (Figure 6a).

Figure 6 Open in figure viewer PowerPoint Distribution of five haplotypes of the GmSALT3 gene in a soybean minicore collection from different ecoregions of China. (a) Five haplotypes in the 172 minicore collection of Chinese soybean landraces and their relationship with salt tolerance. +/−, with or without the insertion in the promoter or exon region. (b) Genetic structure of populations based on data from 30 simple sequence repeat markers distributed on 20 soybean chromosomes. The length of each coloured segment indicates the attribution of ancestry of each accession. The distribution of each haplotype in ecoregions and phenotypes is indicated by red diamonds and green down triangles and is shown above the plot of population structure. NR, northern ecoregion, including the north‐east spring subregion (NESp) and the north spring subregion (NSp); HR, Huang‐Huai region, including the Huang‐Huai spring subregion (HSp) and the Huang‐Huai summer subregion; SR, southern ecoregion, including the south spring subregion (SSp), south summer subregion (SSu) and south autumn subregion (SAu). (c) Geographic distribution of five haplotypes (H1–H5) in three soybean growing ecoregions (NR, HR and SR) in China.

Using this sequence information we developed a set of haplotype‐specific markers to genotype 172 soybean landraces from the Chinese soybean minicore collection (this included the 31 sequenced accessions used above) (Tables S1 and S3). The soybean minicore collection was selected to represent the maximum genetic diversity of Chinese soybean landraces and has been successfully used to study natural variation in the domestication‐related gene GmTfl1 (Tian et al., 2010). The salt sensitivity of these landraces was screened four times from 2009 to 2011 (Table S2). Of the 172 representative accessions, 73 out of the 76 salt‐tolerant accessions shared H1; 14 salt‐sensitive and 1 salt‐tolerant landraces contained H2; 5 salt‐sensitive accessions contained H3; 25 salt‐sensitive and 3 salt‐tolerant accessions contained H4; and 45 salt‐sensitive accessions contained H5. Three heterozygous landraces were excluded from further analyses. We analysed the geographical distribution of the five haplotypes represented in this minicore collection. Haplotype 1 was observed in the three main growing ecoregions (Tian et al., 2010), and was mostly distributed in the northern eco‐region (NR) and the Huang‐Huai ecoregion (HR); most H2‐containing accessions originated from the northern eco‐region (NR), H3 was distributed mainly in the southern eco‐region (SR); H4 and H5 were observed in the SR and Huang‐Huai ecoregion (HR), but were mainly found in SR (Figure 6b,c).

To further examine the relationship between salt tolerance and the GmSALT3/Gmsalt3 alleles, we sequenced 22 wild soybean (Glycine soja Sieb. et Zucc.) that differed in their salt tolerance. Four of the haplotypes observed in soybean landraces were found in wild soybean but not H2, and four new haplotypes (H6–H9) were found. Compared with H1, H6 had a 21‐bp deletion in exon 5, and H8 and H9 had three different non‐synonymous SNPs from that of H3 (Figure 7a). Haplotype‐specific markers were used to genotype the 57 wild soybean (including the 22 that we had sequenced). As we observed for the soybean landraces, H1 was mainly in salt‐tolerant germplasm (Figure S7a,b). In addition the two wild accessions containing H7 were both salt tolerant, whilst the other haplotypes were predominantly found in salt‐sensitive wild accessions (Figure S7a,b, Table S4). In wild soybean, H1 and H7 were mainly distributed in the NR and HR regions, H3 was seen only in the SR, H4 was present in both NR and SR regions and H5 and wild soybean‐specific H6, H8 and H9 haplotypes were mainly present in SR. This suggests that the distribution of haplotypes in landraces and wild soybean plants were similar (Figure 7b). As annotated in Figure 7(b), most of the saline soil in China is distributed in four main areas: the eastern coast of China including Jiangsu, Shandong, Hebei and Liaoning provinces; the North China plain, the north‐east Songnen plain and the inland region of north‐east China. There are also isolated saline fields with a scattered distribution south of the Yangtze River, within Zhejiang, Fujian and Guangdong provinces (Wang, 1993; Tang and Qiao, 2008; Yang, 2008). The coincidence of salt‐affected soils and the salt‐tolerant H1 and H7 haplotypes indicates that these alleles are likely to be a major selection factor determining the distribution and utilization of soybean especially on saline soils. Consistent with this hypothesis is the significant association observed between those wild soybean accessions that contain H1 and H7 and their proximity to the known saline‐affected regions compared with those that contain the other haplotypes (Figure S7c) (Wilcoxon–Mann–Whitney test, P‐value 0.000001).

Figure 7 Open in figure viewer PowerPoint Variation analysis of GmSALT3 in landraces and wild soybean and haplotype distribution. (a) Distribution of DNA polymorphisms in the 540‐bp promoter region and coding region among 31 landraces and 22 wild soybean. Blue and green indicate the nucleotide difference with Tiefeng 8. The asterisk indicates amino acid change. NA, not detected. (b) Geographical distribution of eight haplotypes in wild soybean. The regions of saline‐affected soil were obtained from Wang (1993) and are presented in yellow on the map. (c) Phylogenetic tree of 31 landraces and 22 wild soybean based on the polymorphic sites of the GmSALT3 coding region. Three major groups were identified, one is mainly for the tolerance alleles H1 and H7, the second is for H2, another is mainly for the sensitive alleles H3–H5, H8 and H9. (d) Haplotype network of the GmSALT3 coding region in landraces and wild soybean. Circle size is proportional to the number of samples within a given haplotype, and black spots represent unobserved, inferred haplotypes. Lines between haplotypes represent mutational steps between alleles. Yellow, landraces; blue, wild soybean.

To seek further evidence that the H1 haplotype has been under selection we analysed the nucleotide diversity of H1–H5 in the landraces within a non‐coding region (1267 bp of intron 2) and found the genetic diversity of this introgenic region in H1 (π = 0.00035) was only 5.5% of that in the sensitive haplotypes H3–H5 (π = 0.00632). Furthermore, we examined the genetic diversity of 18 SNPs within 12 genes on chromosome 3 within 194 kb of the GmSALT3 gene (Figure S8a). The collective genetic diversity in H1 landraces ranged from 0 to 0.1142, with an average of 0.0341, while the diversity in salt‐sensitive haplotypes (H2–H5) ranged from 0.0435 to 0.4999, with an average of 0.3250. In contrast, when we analysed the genetic diversity of randomly selected regions of the genome away from GmSALT3 we found that diversity was similar across all haplotypes, being 0.2428 in H1 and 0.2689 in H2–H5 (Figure S8b). Taken together, we conclude that the low genetic diversity of salt‐tolerant haplotypes is likely to be a result of severe selection pressure.

To examine the relatedness of the nine haplotypes and to determine which haplotype is the likely ancestral allele in soybean we again compared their sequence, their geographical distribution and their salt‐tolerance phenotypes. The salt‐tolerant H1 is the most frequently found haplotype in both wild soybean and landraces, and it has the widest geographical range. The other salt‐tolerant haplotype, H7, which was found only twice and only in wild soybean is identical to H1 except for one non‐synonymous SNP, whilst H2 was identical to H7 except for the 3.78‐kb copia retrotransposon insertion (Figure 7a). Furthermore, both H2 and H7 were found predominantly in the NR (Figures 6c and 7b). By comparing nucleotide polymorphisms within the 540‐bp promoter sequences we observed that H1, H2 and H7 shared similar variation and H2 and H7 shared exactly the same sequence, as did H3, H4 and H8, and H5, H6 and H9 (Figure 7a). Collectively, this suggests that H2 was derived from H7 during or after domestication, and H7 was derived from H1. The other six salt‐sensitive haplotypes were separated from H1 and H7 by a series of mutation events but shared a fixed variation of a 148/150‐bp and 4‐bp insertion at the promoter region, indicating that these variations come from a common haplotype that was not detected in the 57 wild soybean used in this study (Figure 7c,d). To explore how these fixed variations in the promoter region affect gene expression we examined GmSALT3 transcript abundance in the soybean cultivars Mayibao (containing H3) and Jinshanchamoshidou (containing H4) and found that the expression of GmSALT3 in the roots of these two soybean cultivars was significantly lower than that of Tiefeng 8 under both control and salt‐stressed conditions (Figure S8c,d).

To examine if the relationship between salt tolerance and the salt‐tolerant alleles held in germplasm introduced from the United States we examined the genotype and phenotype of 12 further soybean accessions including the sequenced Williams 82 (Schmutz et al., 2010). Several formerly reported salt‐tolerant accessions, including Lee 68, Forrest and Hartwig, had the H1 haplotype, whilst the sensitive accessions Clark and Williams 82 contained the H2 haplotype (Table S2) (Lee et al., 2008; Valencia et al., 2008).