in Soybean and Greatly Increases the Grain Yield in Saline Field Conditions cheap real jordans
Ncl Synchronously Regulates Na+, K+, and Cl in Soybean and Greatly Increases the Grain Yield in Saline Field ConditionsTuyen Duc Do1, 2 n1, Huatao Chen1, 3 n1, Vu Thi Thu Hien1, 4, Aladdin Hamwieh1, 5, Tetsuya Yamada6, Tadashi Sato7, Yongliang Yan8, Hua Cong8, Mariko Shono1, Kazuhiro Suenaga1 Donghe Xu1Scientific Reports 6, Article number: 19147 (2016)doi:10.1038/srep19147Download CitationPlant breedingPlant geneticsAbstractSalt stress inhibits soybean growth and reduces gain yield. Genetic improvement of salt tolerance is essential for sustainable soybean production in saline areas. In this study, we isolated a gene (Ncl) that could synchronously regulate the transport and accumulation of Na+, K+, and Cl from a Brazilian soybean cultivar FT Abyara using map based cloning strategy. Higher expression of the salt tolerance gene Ncl in the root resulted in lower accumulations of Na+, K+, and Cl in the shoot under salt stress. Transfer of Ncl with the Agrobacterium mediated transformation method into a soybean cultivar Kariyutaka significantly enhanced its salt tolerance. Introgression of the tolerance allele into soybean cultivar Jackson, using DNA marker assisted selection (MAS), produced an improved salt tolerance line. Ncl could increase soybean grain yield by 3.6 5.5 times in saline field conditions. Using Ncl in soybean breeding through gene transfer or MAS would contribute to sustainable soybean production in saline prone areas. polo outlet
IntroductionSoybean [Glycine max (L.) Merr.] is the world's primary crop source for protein and oil. Soybean 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. Soybean is generally regarded as a salt sensitive crop compared with other major crops such as wheat, rice, and cotton. Salt stress inhibits soybean germination and plant growth1,2, nodule formation3, and seed yield4.
Genetic variation for salt tolerance has been described in soybean1,4,5,6, The salt tolerance of partial soybean germplasm conserved in the GRIN, USDA has been evaluated. Salt tolerance variation was also observed in wild soybean [Glycine soja Sieb. Zucc.] and some wild soybean accessions were identified to be salt tolerant7,8,9,10,11,12. The high level of variation in soybean germplasm, including wild and cultivated soybean species, suggest that genetic improvement of salt tolerance is feasible. coach factory outlet online
The heredity of salt tolerance in soybean was previously analyzed as a quality trait using the classical genetics approach13,14, and the gene symbols Ncl and ncl were proposed as the dominant for tolerance and the recessive for sensitive, respectively13. The recent development of soybean molecular maps provides a promising approach in identifying genes associated with traits of interest in soybean15,16. Several quantitative trait loci (QTLs) for salt tolerance were reported in different germplasms8,10,17,18,19. Of these, a major QTL for salt tolerance was constantly detected on soybean chromosome 3 (Linkage group N) in different populations8,10,17,19. The QTL on chromosome 3 is likely to be the Ncl locus based on pedigree tracing17. The salt tolerance QTL, in which the tolerant allele was from a wild soybean accession PI 483463, was recently mapped within a 658 kb region between SSR03_1335 and SSR_1359 on chromosome 310. The 658 kb region contained 80 annotated genes, including two genes (Glyma03g32890 and Glyma03g32900) belonging to the sodium/hydrogen exchanger family. Using the whole genome sequencing approach, a candidate causal gene, GmCHX1, a counterpart of Glyma03g32900 in Williams 82, was identified in a wild soybean accession W0512. Glyma03g32900 (GmSALT3), which associated with limiting the accumulation of sodium ion, was also identified as a candidate causal gene underlying the QTL on chromosome 3 from a Chinese commercial soybean cultivar, Tiefeng 8, by using fine mapping method20. coach online outlet
Functional analyses of sodium (Na+) and potassium (K+) transporters have been extensively conducted because of their roles in both nutrition and salt tolerance21,22,23,24,25,26,27. Several candidates for the cation antiporter associated with salt tolerance such as GmCHX112, GmSALT320, GmCAX128, GmNHX129, GmPIP30, and GmHKT131 have been reported in soybean. In contrast, mechanisms of chloride (Cl) transport in plants are poorly understood, despite the importance of minimizing Cl toxicity for salt tolerance. Control of Cl transport and Cl "exclusion" from shoots is correlated with salt tolerance in soybean1,7,11,13,32 and other legumes Trifolium33, Medicago34, and Lotus35 as well as in other crops, such as wheat36; however, no gene that regulates the transport and accumulation of Cl in soybean has been reported. michael kors outlet
Map based cloning or positional cloning strategies have been successfully used in the isolation of genes underlying QTLs of environmental stress tolerant traits such as salt25, submergence37, and phosphorus deficiency38 tolerances in crops. In this study, we isolated a QTL for salt tolerance using the map based cloning strategy from a from a Brazilian soybean cultivar FT Abyara to facilitate its use in breeding. Our results showed that the salt tolerance gene could synchronously regulate the transport and accumulation of Na+, K+, and Cl in soybean. The near isogenic lines (NILs) carrying the tolerant allele of the QTL could increase soybean grain yield by 3.6 5.5 times in saline field conditions. real cheap jordans
ResultsMap based cloning of the salt tolerance QTLTo identify the gene that conditioned the salt tolerance QTL, we initially conducted fine mapping using 1,053 plants derived from 6 F8 residual heterozygous plants, and narrowed down the QTL to a 58.8 kb region between simple sequence repeat (SSR) markers BARCSOYSSR_03_1342 and BARCSOYSSR_03_1338 (Fig. 1a). However, this genomic region was not enough for identification of the causal gene underlying the QTL because there were seven predicted genes within this region based on the Glyma 1.0 soybean gene annotation database of the Williams 82 genome sequence39, including two genes (Glyma03g32890 and Glyma03g32900) belonging to the sodium/hydrogen exchanger family. Next, a segregation population consisting of 5,828 plants derived from F9 residual heterozygous plants was used for screening recombination between BARCSOYSSR_03_1342 and BARCSOYSSR_03_1338. The recombination screen facilitated in the selection of 29 plants with homozygous genotype at one marker and heterozygous genotype at the other marker. The selected plants were self pollinated for one more generation to generate fixed recombination lines. Salt tolerance evaluation and further genotyping of the recombination lines using newly developed DNA markers delimited the QTL to a 16.6 kb interval between SSR markers SSR25.8 and CAPS42.4. Only one predicted gene, Glyma03g32900, existed within the 16.6 kb region (Fig. 1a). RT PCR analysis showed that the level of transcription of Glyma03g32900 was higher in the salt tolerant line NILs18 T than in the salt sensitive line NILs18 S (Fig. 1b). Glyma03g32900 was thus determined as the causal gene underlying the salt tolerance QTL, and was named as Ncl followed Abel (1969)13 (previously designated as qNaCl3).
Figure 1: Map based cloning and expression of the salt tolerance gene Ncl.(a) Fine mapping delimits Ncl to a 16.6 kp region between SSR25.8 and CAPS42.4 on chromosome 3. amarker position (kb), bmarker position (bp). Red and blue bars represent homozygous chromosome segments for tolerance and sensitivity, respectively. Na+, K+, and Cl leaf contents (n=3) and shoot dry weight (n=5 8) for each recombinant line after a treatment with 100mM NaCl for approximately three weeks in a hydroponic condition. significant difference at Pb) Expression of Ncl analyzed by semi quantitative RT PCR in the roots at one and three days after treatment with 100mM and 0mM (Control) NaCl in a hydroponic condition. The actin gene was used as a control.
A Ty1/copia type retrotransposon might cause loss of function of Ncl in soybeanUsing the 3 Full rapid amplification of cDNA ends (RACE) and the 5 Full RACE methods, full length cDNAs of the tolerant and the sensitive alleles were determined from the salt tolerant line NILs18 T and the sensitive line NILs18 S, respectively. On the basis of the positions of start and stop codons, the coding sequence (CDS) of NILs18 T was determined to be 2,436bp in length and encodes 811 amino acid residues; the CDS of NILs18 S was 1,131bp in length and encodes 376 amino acid residues. Comparison of genomic and cDNA sequences showed that Ncl harbored five CDSs in the salt tolerant line NILs18 T, whereas three CDSs were detected in the sensitive line NILs18 S (Supplementary Fig. S1a online). The NILs18 S possessed a 3.8 kb insertion compared with the tolerant line NILs18 T. Because there were long repeated sequences within the 3.8 kb fragment, which suggested that the element belongs to a retrotransposon family, we conducted a BLAST analysis using a soybean transposable element database. We found that the 3.8 kb fragment is probably a Ty1/copia type retrotransposon. Alignment of the full length cDNA sequences for NILs18 T and NILs18 S showed a point mutant at the 5 untranslated region (UTR). In the 3 end of the sensitive line (NILs18 S) cDNA, two polyadenylation signals of AATAAA were detected at 38bp and 60bp upstream of the poly (A) tail (Supplementary Fig. S2 online). The polyadenylation signal, which was introduced by the Ty1/copia type retrotransposon, may be responsible for the transcription termination of Ncl in the sensitive line.
The genotype of the soybean root, but not shoot, determines salt toleranceIn this study, Ncl expression was significantly higher in the root tissues compared with the other plant parts (data not shown). To understand the role of the root system in salt tolerance, we grafted the arable part of the salt tolerant line NILs25 T to the root of the sensitive line NILs25 S, or in reverse, to produce two kinds of graft hybrids: NILs25 T (shoot)/NILs25 S (root) and NILs25 S (shoot)/NILs25 T (root). Salt tolerance evaluation for the two kinds graft hybrids as well as the NILs pair NILs25 T and NILs25 S with 100mM NaCl in a hydroponic condition revealed that NILs25 T and the graft hybrid NILs25 S/NILs25 T showed higher salt tolerance than NILs25 S and the graft hybrid NILs25 T/NILs25 S in terms of leaf SPAD value, shoot dry weight, and Na+, K+, and Cl concentration in the shoots, indicating that the root genotype, but not the shoot genotype, determines salt tolerance in soybean (Supplementary Fig. S3 and S4 online). coach diaper bag
Ncl synchronously regulates the accumulation of Na+, K+, and Cl in soybean leavesBLAST analysis showed that the Ncl was homologous to the Na+/H+ antiporter gene family (Supplementary Fig. S1b online). It had regulatory functions for Na+ and K+ transport in soybean plant. However, our analysis observed that Ncl also regulated Cl accumulation. The salt tolerant lines showed lower Na+, K+, and Cl contents in the leaves than the sensitive lines under salt stress conditions (Fig. 1a; Supplementary Fig. S4 online). Another interesting result was that the Na+ and K+ distributions in soybean plants (roots and shoots) were different from that of Cl under salt stress conditions. In comparison with the salt sensitive lines, the salt tolerant line accumulated lower Na+ and K+ concentrations in both shoots (leaves and stems) and roots. In contrast, the salt tolerant line had lower Cl concentrations in shoots (leaves and stems) and higher Cl concentration in root (Supplementary Fig. S4 online). We also tested the three sets of NILs under the stress condition of 100mM KCl. No significant differences in leaf Cl content were observed between the tolerant and sensitive lines (Supplementary Fig. S5a online). Both tolerant and sensitive lines showed chlorosis symptom under 100mM KCl stress condition (Supplementary Fig. S5b online). Ncl functioned like the cation chloride cotransporter (CCC) genes that were identified in other plants. However, Ncl showed a very low similarity to these putative CCC genes identified in other plant species (Supplementary Fig. S1b online). cheap jordans for sale
Overexpression of Ncl resulted in improved salt tolerance in transgenic soybeanWe transformed Ncl full length cDNA driven with 35S promoter (35S:Ncl) into a Japanese soybean cultivar Kariyutaka using the Agrobacterium mediated transformation method. We used Kariyutaka for gene transformation because it is highly amenable to Agrobacterium mediated transformation40 and is a salt sensitive cultivar with a relatively low level of expression of Ncl and the same gene structure as the sensitive line NILs 18S (this study). We investigated four T2 transgenic lines (54 1 1, 34 2 7, 20 1 4, and 16 1 8), which were derived from independent explants, for their expression of Ncl and salt tolerance performances. All the four T2 lines showed significantly higher levels of expression of Ncl compared with that of the wild type Kariyutaka and a 35S:GFP transgenic line that was used as control, either in the salt stress of 100mM NaCl or in the control condition (Fig. 2a; Supplementary Fig. S6a online). A close association between expression of Ncl and salt tolerance was observed (Fig. 2b e). The 20 1 4 line, which was single copy number for Ncl (Supplementary Fig. S6b online), showed the highest expression level in the four transgenic lines. As expected, the 20 1 4 line showed the highest salt tolerance in terms of leaf SPAD value, shoot dry weight, and ions (Na+, K+, and Cl) concentration in leaves. The results generated by the soybean transgenic line confirmed the function of the Ncl gene in relation to soybean salt tolerance.
Figure 2: Overexpression of Ncl in transgenic lines enhanced salt tolerance.(a) Real time quantitative RT PCR analysis of Ncl expression levels in the transgenic soybean lines and control materials after treatments with 100mM NaCl for 24 hours in a hydroponic condition. 54 1 1, 34 2 7, 20 1 4, and 16 1 8 are T2 35S:Ncl transgenic lines. Kariyutaka: wild type soybean cultivar. GFP is 35S:GFP transgenic line (T6). NILs18 T and NILs18 S are tolerant and sensitive near isogenic lines. Values represent means from three biological replicates. (b,c) Leaf SPAD values and shoot dry weight for the transgenic lines after treatments with 100mM NaCl for approximately three weeks in a hydroponic condition. (d) Na+, K+, and Cl leaf contents for the transgenic lines after treatments with 100mM NaCl and 0mM NaCl (Control) for approximately three weeks in a hydroponic condition. (e) Comparison of salt tolerance between 35S:Ncl plants and null plants of the T2 transgenic line 20 1 4. indicate significant difference at P polo outlet online
Expression of Ncl was closely associated with salt tolerance in 123 soybean germplasms from different countriesTo understand the function of Ncl on salt tolerance in different genetic backgrounds, we analyzed the relationship between Ncl expression level and salt tolerance in 123 soybean germplasms, including the three sets of NILs for salt tolerance and seven wild soybean accessions. The cultivated soybean germplasms were collected from nine countries (Supplementary Table S2 online). The expression levels of Ncl showed a close correlation with salt tolerance in terms of leaf SPAD values (r=0.7214, Pr=0.7476, PSupplementary Fig. S7 online), despites the fact that the mRNA level and protein level were not always associated. This result indicated that Ncl was effective in different genetic backgrounds. Other genes need to be considered to explain the difference of expression level of Ncl among the accession that harbored Ncl. coachoutlet.com
Introgression of Ncl into the salt sensitive cultivar Jackson by MASTo determine the usefulness of Ncl in soybean breeding, we conducted introgression of the salt tolerance allele identified in a wild soybean8 into a salt sensitive variety, Jackson (PI 548657), through continuous backcross, followed by MAS using primers Satt339, SSR222042, and SSR112166 in each generation. A BC4F2 plant, which was heterozygous for the three SSR markers, was self pollinated to generate a salt tolerant homozygous line BC4F3 J1T and a sensitive homozygous line BC4F3 J1S. Salt tolerance evaluation with 100mM NaCl in a hydroponic condition showed that BC4F3 J1T had significant higher leaf SPAD values and shoot dry weight, and lower leaf ion (Na+, K+, and Cl) contents than BC4F3 J1S as well as Jackson. In contrast, no significant differences in leaf SPAD value, shoot dry weight, and leaf ion (Na+, K+, and Cl) content in the control condition were observed between BC4F3 J1T and BC4F3 J1S (Fig. 3a d). This result demonstrated that the DNA markers around Ncl could be used for introgression of Ncl into a salt sensitive cultivar for developing of a soybean variety for high salt tolerance.
Figure 3Effect of Ncl on salt tolerance in terms of leave SPAD value (a), shoot dry weight (b), and ion (Na+, K+, and Cl leaf contents (c) in BC4F3 lines developed by MAS by introducing the tolerance allele from a wild soybean accession JWS156 1 into Jackson. BC4F3 J1T and BC4F3 J1S was derived from progeny of self pollination of a BC4F2 plant, which was heterozygous in the Ncl region. (d) Performance of BC4F3 J1T and BC4F3 J1S after treatment with 100mM NaCl for approximately three weeks in a hydroponic condition. (n=11). indicate significant difference at P michael kors canada
The Ncl gene could increase soybean grain yield 3.6 5.5 times in saline field conditionsTo determine the effect of Ncl in a salt stress field condition and its potential in soybean breeding for salt tolerance, we evaluated the NILs for salt tolerance in a salt stress field condition. The field experiments were conducted for three years. In 2009 and 2011, the three sets of NILs, including a salt sensitive cultivar, Tachiyutaka, were tested. As a result, all the lines with the Ncl allele showed a significantly higher yield than the lines with the sensitive allele (Fig. 4a e; Supplementary Fig. S8 online). In the 2011 field experiment, the average yield of the three tolerant lines (NILs18 T, NILs25 T, and NILs72 T) was 2.383 t/h, whereas the average yield of the three sensitive lines (NILs18 S, NILs25 S, and NILs72 S) was 0.437 t/h, increasing the grain yield by 5.5 times in a saline field condition (Fig. 4c). In 2012, we tested another six fixed lines derived from an F8 residual heterozygous line for the QTL region. These six lines have the same genetic background but different chromosome length for the salt tolerance QTL region. Three (N18 39, N18 99, and N18 122) of these lines were salt tolerant, whereas three lines (N18 9, N18 61, N18 180) were salt sensitive. The results of field experiment showed that the average yield for the three tolerant lines was 2.973 t/h, whereas that for the three sensitive lines was 0.828 t/h, indicating an increase in the grain yield by 3.6 fold in a saline field condition (Fig. 4d). In contrast, no significant differences in grain yield were observed between the tolerant and sensitive NILs in a control condition (Fig. 4c,d). Salt stress reduced the grain yield for all the lines by 55.8% in 2011 and 44.0% in 2012. For the lines with the Ncl allele, the yield decreased on average by 26.7% in 2011 and 29.5% in 2012, whereas those without the Ncl allele were 86.0% in 2011 and 76.5% in 2012. These results clearly indicated that Ncl contributed to achieving sustainable soybean production in a salt stress condition. cheap real jordans
Figure 4: Yield performances of lines carrying Ncl in a saline field condition in Miyagi Prefecture, Japan.(a) Top view of NILs25 S and NILs25 T grown in a salt stress field in 2009. (b) Top view of N18 61 and N18 99 grown in a salt stress field in 2012. (c) Grain yield result of the three sets of NILs in 2011 field test. (d) Grain yield result of six recombinant lines derived coach purses outlet
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