Identification of <Emphasis Type="Italic">GmSALT3</Emphasis> haplotypes and development of molecular markers based on their diversity associated with salt tolerance in soybean

Soybean [Glycine max (L.) Merr.] is a major agricultural crop and generally known as a salt-sensitive crop. In a previous study, GmSALT3 was identified as a salt tolerance gene in soybean, and its nine haplotypes (H1 to H9) were reported in Chinese soybean accessions. In the present study, we aimed to identify new haplotypes of GmSALT3 in soybean accessions and to develop molecular markers for selection of salt-tolerant and -sensitive accessions. To do so, we examined genomic variations in the GmSALT3 coding region of 216 accessions of G. max and G. soja from Korea, China, and Japan. As a result, 40 different haplotypes, including three known haplotypes (H1, H2, and H5), were identified. Performing salt tolerance tests for the haplotypes, we were able to classify them into salt-tolerant (8) and salt-sensitive (32) categories. We also found that more variations in the haplotype composition of G. soja accessions exist than in that of G. max accessions. Quantitative expression analysis showed that almost all of the salt-tolerant haplotypes had much higher levels of GmSALT3 expression than the salt-sensitive haplotypes did. Finally, we developed molecular markers and applied them to screen salt tolerance of soybean accessions. The molecular markers performed well with an accuracy of 98.8% in identifying phenotypes of soybean accessions.     

Photosynthesis and yield traits in different soybean lines in response to salt stress

To evaluate utility of different salt-tolerant lines, three soybean lines with different resistance to salt were planted in the field under control and salt-stress conditions for two years. The results showed that net photosynthetic rate (P_N) was significantly different among lines at the anthesis stage and decreased on average by 13.6-34.1% under conditions of salt stress. The stomatal conductance was a primary limiting factor for the reduction of P_N under salt stress. Meanwhile, the grain yield (GY) decreased on average by 14.0-35.3% among lines under salt stress. The salt-tolerant lines S111-9 and S113-6 showed higher P_N and GY under salt stress in comparison with the salt-sensitive cultivar Melrose. Regression analysis indicated that there was extremely significantly positive correlation between GY and P_N under field conditions. Therefore, P_N might be used as a physiological index for field resistance of soybean to salt stress.     

A <Emphasis Type="Italic">bean common mosaic virus</Emphasis> (BCMV)-resistance gene is fine-mapped to the same region as <Emphasis Type="Italic">Rsv1</Emphasis>-<Emphasis Type="Italic">h</Emphasis> in the soybean cultivar Suweon 97

Key message

In the soybean cultivar Suweon 97, BCMV-resistance gene was fine-mapped to a 58.1-kb region co-localizing with the Soybean mosaic virus (SMV)-resistance gene, Rsv1-h raising a possibility that the same gene is utilized against both viral pathogens.

Abstract

Certain soybean cultivars exhibit resistance against soybean mosaic virus (SMV) or bean common mosaic virus (BCMV). Although several SMV-resistance loci have been reported, the understanding of the mechanism underlying BCMV resistance in soybean is limited. Here, by crossing a resistant cultivar Suweon 97 with a susceptible cultivar Williams 82 and inoculating 220 F₂ individuals with a BCMV strain (HZZB011), we observed a 3:1 (resistant/susceptible) segregation ratio, suggesting that Suweon 97 possesses a single dominant resistance gene against BCMV. By performing bulked segregant analysis with 186 polymorphic simple sequence repeat (SSR) markers across the genome, the resistance gene was determined to be linked with marker BARSOYSSR_13_1109. Examining the genotypes of nearby SSR markers on all 220 F₂ individuals then narrowed down the gene between markers BARSOYSSR_13_1109 and BARSOYSSR_13_1122. Furthermore, 14 previously established F_2:3 lines showing crossovers between the two markers were assayed for their phenotypes upon BCMV inoculation. By developing six more SNP (single nucleotide polymorphism) markers, the resistance gene was finally delimited to a 58.1-kb interval flanked by BARSOYSSR_13_1114 and SNP-49. Five genes were annotated in this interval of the Williams 82 genome, including a characteristic coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR, CNL)-type of resistance gene, Glyma13g184800. Coincidentally, the SMV-resistance allele Rsv1-h was previously mapped to almost the same region, thereby suggesting that soybean Suweon 97 likely relies on the same CNL-type R gene to resist both viral pathogens.

     

Fine-mapping and identification of a novel locus <Emphasis Type="Italic">Rsc15</Emphasis> underlying soybean resistance to <Emphasis Type="Italic">Soybean mosaic virus</Emphasis>

Key message

Rsc15, a novel locus underlying soybean resistance to SMV, was fine mapped to a 95-kb region on chromosome 6. The Rsc15- mediated resistance is likely attributed to the gene GmPEX14 , the relative expression of which was highly correlated with the accumulation of H ₂ O ₂ along with the activities of POD and CAT during the early stages of SMV infection in RN-9.

Abstract

Soybean mosaic virus (SMV) causes severe yield losses and seed quality deterioration in soybean [Glycine max (L.) Merr.] worldwide. A series of single dominant SMV resistance genes have been identified on respective soybean chromosomes 2, 13 and 14, while one novel locus, Rsc15, underlying resistance to the virulent SMV strain SC15 from soybean cultivar RN-9 has been recently mapped to a 14.6-cM region on chromosome 6. However, candidate gene has not yet been identified within this region. In the present study, we aimed to fine map the Rsc15 region and identify candidate gene(s) for this invaluable locus. High-resolution fine-mapping revealed that the Rsc15 gene was located in a 95-kb genomic region which was flanked by the two simple sequence repeat (SSR) markers SSR_06_17 and BARCSOYSSR_06_0835. Allelic sequence comparison and expression profile analysis of candidate genes inferred that the gene Glyma.06g182600 (designated as GmPEX14) was the best candidate gene attributing for the resistance of Rsc15, and that genes encoding receptor-like kinase (RLK) (i.e., Glyma.06g175100 and Glyma.06g184400) and serine/threonine kinase (STK) (i.e., Glyma.06g182900 and Glyma.06g183500) were also potential candidates. High correlations were established between the relative expression level of GmPEX14 and the hydrogen peroxide (H₂O₂) concentration and activities of catalase (CAT) and peroxidase (POD) during the early stages of SMV-SC15 infection in RN-9. The results of the present study will be useful in marker-assisted breeding for SMV resistance and will lead to further understanding of the molecular mechanisms of host resistance against SMV.

     

RNAi-mediated <Emphasis Type="Italic">Soybean mosaic virus</Emphasis> (SMV) resistance of a Korean Soybean cultivar

Soybean [Glycine max (L.) Merr.] is an important crop for vegetable oil production, and is a major protein source worldwide. Because of its importance as a crop, genetic transformation has been used extensively to improve its valuable traits. Soybean mosaic virus (SMV) is one of the most well-known viral diseases affecting soybean. Transgenic soybean plants with improved resistance to SMV were produced by introducing HC-Pro coding sequences within RNA interference (RNAi) inducing hairpin construct via Agrobacterium-mediated transformation. During an experiment to confirm the response of transgenic plants (T₂) to SMV infection, no T₂ plants from lines #2 (31/31), #5 (35/35) or #6 (37/37) exhibited any SMV symptoms, indicating strong viral resistance (R), whereas NT (non-transgenic wild type) plants and those from lines #1, #3 and #4 exhibited mild mosaic (mM) or mosaic (M) symptoms. The northern blot analysis showed that three resistant lines (#2, #5 and #6) did not show the detection of viral RNA accumulation while NT, EV (transformed with empty vector carrying only Bar) and lines #1, #3 and #4 plants were detected. T₃ seeds from SMV-inoculated T₂ plants were harvested and checked for changes in seed morphology due to viral infection. T₃ seeds of lines #2, #5 and #6 were clear and seed coat mottling was not present, which is indicative of SMV resistance. RT-PCR and quantitative real-time PCR showed that T₃ seeds from the SMV-resistant lines #2, #5 and #6 did not exhibit any detection of viral RNA accumulation (HC-Pro, CP and CI), while the viral RNA accumulation was detected in SMV-susceptible lines #1, #3 and #4 plants. During the greenhouse test for viral resistance and yield components, T₃ plants from the SMV-inoculated transgenic lines #2, #5 and #6 showed viral resistance (R) and exhibited a more favorable average plant height, number of nodes per plant, number of branches per plant, number of pods per plant and total seed weight with statistical significance during strong artificial SMV infection than did other plant lines. In particular, the SMV-resistant line #2 exhibited superior average plant height, pod number and total seed weight with highly significance. According to our results, RNAi induced by the hairpin construct of the SMV HC-Pro sequence effectively confers much stronger viral resistance than did the methods used during previous trials, and has the potential to increase yields significantly. Because of its efficiency, the induction of RNAi-mediated resistance will likely be used more frequently as part of the genetic engineering of plants for crop improvement.     

A novel <Emphasis Type="Italic">TaSST</Emphasis> gene from wheat contributes to enhanced resistance to salt stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

In this research, through the analyzing of the Triticum aestivum salt-tolerant mutant gene expression profile, under salt stress. A brand new gene with unknown functions induced by salt was cloned. The cloned gene was named Triticum aestivum salt stress protein (TaSST). GenBank accession number of TaSST is ACH97119. Quantitative polymerase chain reaction (qPCR) results exhibited that the expression TaSST was induced by salt, abscisic acid (ABA), and polyethylene glycol (PEG). TaSST could improve salt tolerance of Arabidopsis-overexpressed TaSST. After salt stress, physiological indexes of transgenic Arabidopsis were better compared with WT (wild-type) plants. TaSST was mainly located in the cytomembrane. qPCR analyzed the expression levels of nine tolerance-related genes of Arabidopsis in TaSST-overexpressing Arabidopsis. Results showed that the expression levels of SOS3, SOS2, KIN2, and COR15a significantly increased, whereas the expression of the five other genes showed no obvious change. OsI_01272, the homologous gene of TaSST in rice, was interfered using RNA interference (RNAi) technique. RNAi plants became more sensitive to salt than control plants. Thus, we speculate that TaSST can improve plant salt tolerance.     

Single nucleotide polymorphism markers for rapid detection of the <Emphasis Type="Italic">Rsv4</Emphasis> locus for soybean mosaic virus resistance in diverse germplasm

Soybean mosaic virus (SMV) causes a substantial decrease in soybean yield and reduction of seed quality. The most effective management strategy to control the virus is the deployment of host resistance. Seven SMV strains and three independent multi-allelic loci for SMV resistance have been identified previously. The goal of this research was to detect single nucleotide polymorphisms (SNPs) associated with SMV resistance at the Rsv4 locus. Ten soybean accessions, with confirmed resistance genes, were used for sequencing the candidate gene Glyma.02g121400. Alignment of these sequences revealed three SNPs displaying 100% consistency for genotypes carrying the Rsv4 gene. These SNPs were applied for a rapid screen of diverse soybean germplasm using the Sequenom iPLEX Gold platform, phenotyped with SMV-G1 and G7 strains to determine phenotype and classified into several groups carrying the proposed R-gene. The population of V94-5152 (Rsv4) × Lee 68 (rsv) was screened using novel SNPs to create a genetic map with improved resolution to determine the location of the Rsv4. To observe the recombination frequencies within the population, three additional SNPs on both sides of the Glyma.02g121400 gene were added. A linkage map revealed a distance of 3.6 cM between the Rsv4 locus and the closest SNP, thus shifting the putative Rsv4 region downstream on chromosome 2. With this region, five candidate genes have been proposed. The genomic position of the discovered SNPs, linked to the Rsv4, could increase screening precision and accelerate breeding efforts to develop multi-strain-resistant crops.     

Salt-stress-responsive chloroplast proteins in <Emphasis Type="Italic">Brassica juncea</Emphasis> genotypes with contrasting salt tolerance and their quantitative PCR analysis

Brassica juncea is mainly cultivated in the arid and semi-arid regions of India where its production is significantly affected by soil salinity. Adequate knowledge of the mechanisms underlying the salt tolerance at sub-cellular levels must aid in developing the salt-tolerant plants. A proper functioning of chloroplasts under salinity conditions is highly desirable to maintain crop productivity. The adaptive molecular mechanisms offered by plants at the chloroplast level to cope with salinity stress must be a prime target in developing the salt-tolerant plants. In the present study, we have analyzed differential expression of chloroplast proteins in two Brassica juncea genotypes, Pusa Agrani (salt-sensitive) and CS-54 (salt-tolerant), under the effect of sodium chloride. The chloroplast proteins were isolated and resolved using 2DE, which facilitated identification and quantification of 12 proteins that differed in expression in the salt-tolerant and salt-sensitive genotypes. The identified proteins were related to a variety of chloroplast-associated molecular processes, including oxygen-evolving process, PS I and PS II functioning, Calvin cycle and redox homeostasis. Expression analysis of genes encoding differentially expressed proteins through real time PCR supported our findings with proteomic analysis. The study indicates that modulating the expression of chloroplast proteins associated with stabilization of photosystems and oxidative defence plays imperative roles in adaptation to salt stress.     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

Mapping novel aphid resistance QTL from wild soybean, <Emphasis Type="Italic">Glycine soja</Emphasis> 85-32

Key message

Two novel QTLs conferring aphid resistance were mapped and validated on soybean chromosomes 8 and 16, respectively. Closely linked markers were developed to assist breeding for aphid resistance.

Abstract

Soybean aphid, Aphis glycines Matsumura, is a highly destructive pest for soybean production. E08934, a soybean advanced breeding line derived from the wild soybean Glycine soja 85-32, has shown strong resistance to aphids. To dissect the genetic basis of aphid resistance in E08934, a mapping population (070020) consisting of 140 F₃-derived lines was developed by crossing E08934 with an aphid-susceptible line E00003. This mapping population was evaluated for aphid resistance in a greenhouse trial in 2010 and three field trials in 2009, 2010, and 2011, respectively. The broad-sense heritability across the field trials was 0.84. In the mapping population 070020, two major quantitative trait loci (QTL) were detected as significantly associated with aphid resistance, and designated as Rag6 and Rag3c, respectively. Rag6 was mapped to a 10.5 centiMorgan (cM) interval between markers MSUSNP08-2 and Satt209 on chromosome 8, explaining 19.5–46.4% of the phenotypic variance in different trials. Rag3c was located at a 7.5 cM interval between markers MSUSNP16-10 and Sat_370 on chromosome 16, explaining 12.5–22.9% of the phenotypic variance in different trials. Rag3c had less resistance effect than Rag6 across all the trials. Furthermore, Rag6 and Rag3c were confirmed in two validation populations with different genetic backgrounds. No significant interaction was detected between Rag6 and Rag3c in either the mapping population or the validation populations. Both Rag6 and Rag3c were indicated as conferring antibiosis resistance to aphids by a no-choice test. The new aphid-resistance gene(s) derived from the wild germplasm G. soja 85-32 are valuable in improving soybeans for aphid resistance.

     

Comparative analysis of Histone modifications and DNA methylation at <Emphasis Type="Italic">OsBZ8</Emphasis> locus under salinity stress in IR64 and Nonabokra rice varieties

Rice being an important cereal crop is highly sensitive to salinity stress causing growth retardation and loss in productivity. However, certain rice genotypes like Nonabokra and Pokkali show a high level of tolerance towards salinity stress compared to IR64 variety. This differential response of tolerant varieties towards salinity stress may be a cumulative effect of genetic and epigenetic factors. In this study, we have compared the salinity-induced changes in chromatin modifications at the OsBZ8 locus in salt-tolerant Nonabokra and salt-sensitive IR64 rice varieties. Expression analysis indicates that the OsBZ8 gene is highly induced in Nonabokra plants even in the absence of salt stress, whereas in IR64, the expression significantly increases only during salt stress. Sequence analysis and nucleosomal arrangement within the region ?2000 to +1000 of OsBZ8 gene show no difference between the two rice varieties. However, there was a considerable difference in histone modifications and DNA methylation at the locus between these varieties. In Nonabokra, the upstream region was hyperacetylated at H3K9 and H3K27, and this acetylation did not change during salt stress. However, in IR64, histone acetylation was observed only during salt stress. Moreover, the upstream region of OsBZ8 gene has highly dynamic nucleosome arrangement in Nonabokra, compared to IR64. Furthermore, loss of DNA methylation was observed at OsBZ8 locus in Nonabokra control plants along with low H3K27me3 and high H3K4me3. Control IR64 plants show high DNA methylation and enriched H3K27me3. Collectively these results indicate a significant difference in chromatin modifications between the rice varieties that regulates differential expression of OsBZ8 gene during salt stress.     

Characterization and genetics of multiple soybean aphid biotype resistance in five soybean plant introductions

Key message

Five soybean plant introductions expressed antibiosis resistance to multiple soybean aphid biotypes. Two introductions had resistance genes located in the Rag1, Rag2, and Rag3 regions; one introduction had resistance genes located in the Rag1, Rag2, and rag4 regions; one introduction had resistance genes located in the Rag1 and Rag2 regions; and one introduction had a resistance gene located in the Rag2 region.

Abstract

Soybean aphid (Aphis glycines Matsumura) is the most important soybean [Glycine max (L.) Merr.] insect pest in the USA. The objectives of this study were to characterize the resistance expressed in five plant introductions (PIs) to four soybean aphid biotypes, determine the mode of resistance inheritance, and identify markers associated with genes controlling resistance in these accessions. Five soybean PIs, from an initial set of 3000 PIs, were tested for resistance against soybean aphid biotypes 1, 2, 3, and 4 in choice and no-choice tests. Of these five PIs, PI 587663, PI 587677, and PI 587685 expressed antibiosis against all four biotypes, while PI 587972 and PI 594592 expressed antibiosis against biotypes 1, 2, and 3. F₂ populations derived from PI 587663 and PI 587972 were evaluated for resistance against soybean aphid biotype 1, and populations derived from PIs 587677, 587685, and 594592 were tested against biotype 3. In addition, F_2:3 plants were tested against biotypes 2 and 3. Genomic DNA from F₂ plants was screened with markers linked to Rag1, Rag2, Rag3, and rag4 soybean aphid-resistance genes. Results showed that PI 587663 and PI 594592 each had three genes with variable gene action located in the Rag1, Rag2, and Rag3 regions. PI 587677 had three genes with variable gene action located in the Rag1, Rag2 and rag4 regions. PI 587685 had one dominant gene located in the Rag1 region and an additive gene in the Rag2 region. PI 587972 had one dominant gene located in the Rag2 region controlling antixenosis- or antibiosis-type resistance to soybean aphid biotypes 1, 2, or 3. PIs 587663, 587677, and 587685 also showed antibiosis-type resistance against biotype 4. Information on multi-biotype aphid resistance and resistance gene markers will be useful for improving soybean aphid resistance in commercial soybean cultivars.

     

Salt oversensitivity derived from mutation breeding improves salinity tolerance in barley <Emphasis Type="Italic">via</Emphasis> ion homeostasis

Salt stress imposes a major environmental threat to agriculture, therefore, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any breeding strategy. In the present study, the expression profile of genes involved in ion homeostasis including salt overly sensitive (HvSOS1, HvSOS2, HvSOS3), vacuolar Na⁺/H⁺ antiporter (HvNHX1), and H⁺-ATPase (HVA) along with ion content measurement were investigated in two genotypes of Hordeum vulgare under 300 mM NaCl. The gene expressions were measured in the roots and shoots of a salt-tolerant mutant genotype M4-73-30 and in its wild-type cv. Zarjou by real-time qPCR technique. The critical differences between the salt-tolerant mutant and its wild-type were observed in the expressions of HvSOS1 (105-fold), HvSOS2 (24-fold), HvSOS3 (31-fold), and HVA (202-fold) genes in roots after 6-h exposure to NaCl. The parallel early up-regulation of these genes in root samples of the salt-tolerant mutant genotype indicated induction of Na⁺/H⁺ antiporters activity and Na+ exclusion into apoplast and vacuole. The earlier up-regulation of HvSOS1, HVA, and HvNHX1 genes in shoot of the wild-type genotype corresponded to the relative accumulation of Na⁺ which was not observed in salt-tolerant mutant genotype because of efficient inhibitory role of the root in Na+ transport to the shoot. In conclusion, the lack of similarity in gene expression patterns between the two genotypes with similar genetic background may confirm the hypothesis that mutation breeding could change the ability of salt-tolerant mutant genotype for efficient ion homeostasis via salinity oversensitivity response.     

Salt tolerance conferred by expression of a global regulator IrrE from <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in oilseed rape

As salinity is a major threat to sustainable agriculture worldwide, cultivation of salt-tolerant crops becomes increasingly important. IrrE acts as a global regulator and a general switch for stress resistance in Deinococcus radiodurans. In this study, to determine whether the irrE gene can improve the salt tolerance of Brassica napus, we introduced the irrE gene into B. napus by the Agrobacterium tumefaciens-mediated transformation method. Forty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) analyses confirmed that the irrE gene had integrated into the plant genome. Northern as well as Western blot analyses revealed that the transgene was expressed at various levels in transgenic plants. Analysis for the T₁ progenies derived from four independent transformants showed that irrE had enhanced the salt tolerance of T₁ in the presence of 350 mM NaCl. Furthermore, under salt stress, transgenic plants accumulated more compatible solutes (proline) and a lower level of malondialdehyde (MDA), and they had higher activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). However, agronomic traits were not affected by irrE gene overexpression in the transgenic B. napus plants. This study indicates that the irrE gene can improve the salt tolerance of B. napus and represents a promising candidate for the development of crops with enhanced salt tolerance by genetic engineering.