Salt Tolerance in Soybeans: Focus on Screening Methods and Genetics.

Salinity greatly affects the production of soybeans in arid and semi-arid lands around the world. The responses of soybeans to salt stress at germination, emergence, and other seedling stages have been evaluated in multitudes of studies over the past decades. Considerable salt-tolerant accessions have been identified. The association between salt tolerance responses during early and later growth stages may not be as significant as expected. Genetic analysis has confirmed that salt tolerance is distinctly tied to specific soybean developmental stages. Our understanding of salt tolerance mechanisms in soybeans is increasing due to the identification of key salt tolerance genes. In this review, we focus on the methods of soybean salt tolerance screening, progress in forward genetics, potential mechanisms involved in salt tolerance, and the importance of translating laboratory findings into field experiments via marker-assisted pyramiding or genetic engineering approaches, and ultimately developing salt-tolerant soybean varieties that produce high and stable yields. Progress has been made in the past decades, and new technologies will help mine novel salt tolerance genes and translate the mechanism of salt tolerance into new varieties via effective routes.

Identification of loci controlling adaptation in Chinese soya bean landraces via a combination of conventional and bioclimatic GWAS.

Landraces often contain genetic diversity that has been lost in modern cultivars, including alleles that confer enhanced local adaptation. To comprehensively identify loci associated with adaptive traits in soya bean landraces, for example flowering time, a population of 1938 diverse landraces and 97 accessions of the wild progenitor of cultivated soya bean, Glycine soja was genotyped using tGBS® . Based on 99 085 high-quality SNPs, landraces were classified into three sub-populations which exhibit geographical genetic differentiation. Clustering was inferred from STRUCTURE, principal component analyses and neighbour-joining tree analyses. Using phenotypic data collected at two locations separated by 10 degrees of latitude, 17 trait-associated SNPs (TASs) for flowering time were identified, including a stable locus Chr12:5914898 and previously undetected candidate QTL/genes for flowering time in the vicinity of the previously cloned flowering genes, E1 and E2. Using passport data associated with the collection sites of the landraces, 27 SNPs associated with adaptation to three bioclimatic variables (temperature, daylength, and precipitation) were identified. A series of candidate flowering genes were detected within linkage disequilibrium (LD) blocks surrounding 12 bioclimatic TASs. Nine of these TASs exhibit significant differences in flowering time between alleles within one or more of the three individual sub-populations. Signals of selection during domestication and/or subsequent landrace diversification and adaptation were detected at 38 of the 44 flowering and bioclimatic TASs. Hence, this study lays the groundwork to begin breeding for novel environments predicted to arise following global climate change.

Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean.

Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR, and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at fourfold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops.

Targeted association mapping demonstrating the complex molecular genetics of fatty acid formation in soybean.

BACKGROUND: The relative abundance of five dominant fatty acids (FAs) (palmitic, stearic, oleic, linoleic and linolenic acids) is a major factor determining seed quality in soybean. METHODS: To clarify the currently poorly understood genetic architecture of FAs in soybean, targeted association analysis was conducted in 421 diverse accessions phenotyped in three environments and genotyped using 1536 pre-selected SNPs. RESULTS: The population of 421 soybean accessions displayed significant genetic variation for each FA. Analysis of the molecular data revealed three subpopulations, which reflected a trend depending on latitude of cultivation. A total of 37 significant (p < 0.01) associations with FAs were identified by association mapping analysis. These associations were represented by 33 SNPs (occurring in 32 annotated genes); another four SNPs had a significant association with two different FAs due to pleiotropic interactions. The most significant associations were cross-verified by known genes/QTL or consistency across cultivation year and subpopulations. CONCLUSION: The detected marker-trait associations represent a first important step towards the implementation of molecular-marker-based selection of FA composition with the potential to substantially improve the seed quality of soybean with benefits for human health and for food processing.

Detecting SNPs underlying domestication-related traits in soybean.

BACKGROUND: Cultivated soybean (Glycine max) experienced a severe genetic bottleneck during its domestication and a further loss in diversity during its subsequent selection. Here, a panel of 65 wild (G. soja) and 353 cultivated accessions was genotyped at 552 single-nucleotide polymorphism loci to search for signals of selection during and after domestication. RESULTS: The wild and cultivated populations were well differentiated from one another. Application of the Fst outlier test revealed 64 loci showing evidence for selection. Of these, 35 related to selection during domestication, while the other 29 likely gradually became monomorphic as a result of prolonged selection during post domestication. Two of the SNP locus outliers were associated with testa color. CONCLUSIONS: Identifying genes controlling domestication-related traits is important for maintaining the diversity of crops. SNP locus outliers detected by a combined forward genetics and population genetics approach can provide markers with utility for the conservation of wild accessions and for trait improvement in the cultivated genepool.

De novo assembly of soybean wild relatives for pan-genome analysis of diversity and agronomic traits.

Wild relatives of crops are an important source of genetic diversity for agriculture, but their gene repertoire remains largely unexplored. We report the establishment and analysis of a pan-genome of Glycine soja, the wild relative of cultivated soybean Glycine max, by sequencing and de novo assembly of seven phylogenetically and geographically representative accessions. Intergenomic comparisons identified lineage-specific genes and genes with copy number variation or large-effect mutations, some of which show evidence of positive selection and may contribute to variation of agronomic traits such as biotic resistance, seed composition, flowering and maturity time, organ size and final biomass. Approximately 80% of the pan-genome was present in all seven accessions (core), whereas the rest was dispensable and exhibited greater variation than the core genome, perhaps reflecting a role in adaptation to diverse environments. This work will facilitate the harnessing of untapped genetic diversity from wild soybean for enhancement of elite cultivars.

Biparental resequencing coupled with SNP genotyping of a segregating population offers insights into the landscape of recombination and fixed genomic regions in elite soybean.

Identification of genes underlying agronomic traits is dependent on the segregation of quantitative trait loci (QTL). A popular hypothesis is that elite lines are becoming increasingly similar to each other, resulting in large genomic regions with fixed genes. Here, we resequenced two parental modern elite soybean lines [ZhongHuang13 (ZH) and ZhongPin03-5373 (ZP)] and discovered 794,876 SNPs with reference to the published Williams82 genome. SNPs were distributed unevenly across the chromosomes, with 87.1% of SNPs clustering in 4.9% of the soybean reference genome. Most of the regions with a high density of SNP polymorphisms were located in the chromosome arms. Moreover, seven large regions that were highly similar between parental lines were identified. A GoldenGate SNP genotyping array was designed using 384 SNPs and the 254 recombinant inbred lines (F8) derived from the cross of ZP × ZH were genotyped. We constructed a genetic linkage map using a total of 485 molecular markers, including 313 SNPs from the array, 167 simple sequence repeats (SSRs), 4 expressed sequence tag-derived SSRs, and 1 insertion/deletion marker. The total length of the genetic map was 2594.34 cM, with an average marker spacing of 5.58 cM. Comparing physical and genetic distances, we found 20 hotspot and 14 coldspot regions of recombination. Our results suggest that the technology of resequencing of parental lines coupled with high-throughput SNP genotyping could efficiently bridge the genotyping gap and provide deep insights into the landscape of recombination and fixed genomic regions in biparental segregating populations of soybean with implications for fine mapping of QTL.

Molecular footprints of domestication and improvement in soybean revealed by whole genome re-sequencing.

BACKGROUND: Artificial selection played an important role in the origin of modern Glycine max cultivars from the wild soybean Glycine soja. To elucidate the consequences of artificial selection accompanying the domestication and modern improvement of soybean, 25 new and 30 published whole-genome re-sequencing accessions, which represent wild, domesticated landrace, and Chinese elite soybean populations were analyzed. RESULTS: A total of 5,102,244 single nucleotide polymorphisms (SNPs) and 707,969 insertion/deletions were identified. Among the SNPs detected, 25.5% were not described previously. We found that artificial selection during domestication led to more pronounced reduction in the genetic diversity of soybean than the switch from landraces to elite cultivars. Only a small proportion (2.99%) of the whole genomic regions appear to be affected by artificial selection for preferred agricultural traits. The selection regions were not distributed randomly or uniformly throughout the genome. Instead, clusters of selection hotspots in certain genomic regions were observed. Moreover, a set of candidate genes (4.38% of the total annotated genes) significantly affected by selection underlying soybean domestication and genetic improvement were identified. CONCLUSIONS: Given the uniqueness of the soybean germplasm sequenced, this study drew a clear picture of human-mediated evolution of the soybean genomes. The genomic resources and information provided by this study would also facilitate the discovery of genes/loci underlying agronomically important traits.

A platform for soybean molecular breeding: the utilization of core collections for food security.

Soybean is an important crop not only for human consumption but also for its addition of nitrogen to the soil during crop rotation. China has the largest collection of cultivated soybeans (Glycine max) and wild soybeans (Glycine soja) all over the world. The platform of soybean core, mini core and integrated applied core collections has been developed in the past decade based on systematic researches which included the sampling strategies, statistical methods, phenotypic data and SSR markers. Meanwhile, intergrated applied core collections including accessions with single or integrated favorite traits are being developed in order to meet the demand of soybean breeding. These kinds of core collections provide powerful materials for evaluation of germplasm, identification of trait-specific accessions, gene discovery, allele mining, genomic study, maker development, and molecular breeding. Some successful cases have proved the usefulness and efficiency of this platform. The platform is helpful for enhancing utilization of soybean genetic resources in sustainable crop improvement for food security. The efficient utilization of this platform in the future is relying on accurate phenotyping methods, abundant functional markers, high-throughput genotyping platforms, and effective breeding programs.

DNA sequence polymorphism of the Rhg4 candidate gene conferring resistance to soybean cyst nematode in Chinese domesticated and wild soybeans.

Rhg4 is one of the major resistant genes conferring resistance to soybean cyst nematode races 1, 3 and 4. In order to better understand its sequence diversity among different Chinese soybean populations and the impact of human activities on it, we designed 5 primer sets based on its sequence deposited in Genbank (Genbank accession No. AF506518) to obtain the Rhg4 sequence from 104 Chinese cultivated and wild soybean genotypes, and then analyzed the DNA sequence polymorphism in different Chinese soybean populations. The alignment of Rhg4 sequence included 5,216 nucleotide base pairs. A total of 67 single nucleotide polymorphisms (SNPs) including 59 single base changes and 8 DNA insertion-deletions (InDels) were identified with a SNP frequency of 1/78. Except for a 14-base InDel, there were 29 SNPs in coding regions, and among them, 13 were non-synonymous (9 in functional domains with 1 in a leucine-rich repeats region, 2 in a transmembrane region and 6 in a Ser/Thr kinase domain). The probability of substitution at each site was not the same, there were two hot spots, one was in the 5'-untranslated region between positions 124 and 804, and the other was in the region between positions 2520 and 3733. Sequence diversity analysis among 104 soybean genotypes showed π = 0.00102 and θ = 0.00218 for Rhg4. A domestication bottleneck was found because of lower sequence diversity and 58% unique SNPs loss in landraces compared with Glycine soja. Intensive selection increased the sequence diversity of cultivars, which had higher diversity and more unique SNPs than landraces. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-012-9703-1) contains supplementary material, which is available to authorized users.

Synthesis and degradation of the major allergens in developing and germinating soybean seed.

Gly m Bd 28K, Gly m Bd 30K and Gly m Bd 60K are the major soybean (Glycine max (L.) Merr.) allergens limiting the consumption of a good protein source for sensitive individuals. However, little is known about their temporal-spatial expression during seed development and upon germination. The present data shows that soy allergens accumulated in both the embryonic axes and cotyledon, but expression patterns differed depending on the specific allergen. Allergens accumulated sooner and to a greater level in cotyledons than in embryonic axes. Gly m Bd 28 began at 14 d after flowering, 7 to 14 d earlier than Gly m Bd 30K and Gly m Bd 60K. Comparatively, their degradation was faster and more profound in embryonic axes than in cotyledons. Gly m Bd 60K began to decline at 36 h after imbibition and remained detectable up to 108 h in cotyledons. In contrast, the Glym Bd 60K protein was reduced at 24 h, and eventually disappeared at 96 h . In cotyledons Gly m Bd 28K first declined at 24 h, then increased from 36 h to 48 h, followed by its large reduction at 72 h after seed germination. These findings provide useful information on soy allergen biosynthesis and will help move forward towards developing a hypoallergenic soybean for safer food.

Genetic diversity in domesticated soybean (Glycine max) and its wild progenitor (Glycine soja) for simple sequence repeat and single-nucleotide polymorphism loci.

• The study of genetic diversity between a crop and its wild relatives may yield fundamental insights into evolutionary history and the process of domestication. • In this study, we genotyped a sample of 303 accessions of domesticated soybean (Glycine max) and its wild progenitor Glycine soja with 99 microsatellite markers and 554 single-nucleotide polymorphism (SNP) markers. • The simple sequence repeat (SSR) loci averaged 21.5 alleles per locus and overall Nei's gene diversity of 0.77. The SNPs had substantially lower genetic diversity (0.35) than SSRs. A SSR analyses indicated that G. soja exhibited higher diversity than G. max, but SNPs provided a slightly different snapshot of diversity between the two taxa. For both marker types, the primary division of genetic diversity was between the wild and domesticated accessions. Within taxa, G. max consisted of four geographic regions in China. G. soja formed six subgroups. Genealogical analyses indicated that cultivated soybean tended to form a monophyletic clade with respect to G. soja. • G. soja and G. max represent distinct germplasm pools. Limited evidence of admixture was discovered between these two species. Overall, our analyses are consistent with the origin of G. max from regions along the Yellow River of China.

Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis.

Previous studies have shown that ubiquitination plays important roles in plant abiotic stress responses. In the present study, the ubiquitin-conjugating enzyme gene GmUBC2, a homologue of yeast RAD6, was cloned from soybean and functionally characterized. GmUBC2 was expressed in all tissues in soybean and was up-regulated by drought and salt stress. Arabidopsis plants overexpressing GmUBC2 were more tolerant to salinity and drought stresses compared with the control plants. Through expression analyses of putative downstream genes in the transgenic plants, we found that the expression levels of two ion antiporter genes AtNHX1 and AtCLCa, a key gene involved in the biosynthesis of proline, AtP5CS, and the copper chaperone for superoxide dismutase gene AtCCS, were all increased significantly in the transgenic plants. These results suggest that GmUBC2 is involved in the regulation of ion homeostasis, osmolyte synthesis, and oxidative stress responses. Our results also suggest that modulation of the ubiquitination pathway could be an effective means of improving salt and drought tolerance in plants through genetic engineering.

[Genetic variation of SNP loci based on candidate gene for resistance to soybean cyst nematode].

For clarifying the difference of genetic diversity and linkage disequilibrium (LD) level between cultivated (Glycine max (L.) Merr.) and annual wild soybean (Glycine soja Sieb. & Zucc.), genetic variation pattern of 8 SNP loci developed from soybean cyst nematode resistance candidate genes rhg1 and Rhg4 in soybean germplasm were analyzed. The results indicated that G. max population, consisted of cultivated soybean mini-core collection and modern cultivars, had a higher LD levels (R2 value is 0.216) than G. soja population. Since 100% of pairwise loci within a gene and 16.6% of pairwise loci between genes were significant in G. max population, two specific LD regions were formed for each gene. A total of 46 haplotypes were detected in 363 soybean germplasm. The population of G. soja had less number of haplotypes and higher haplotype diversity than the population of G. max. Among the 31 population-specific haplotypes, 15 haplotypes were specific for G. soja population. In addition, the frequency of two major predominant haplotypes (Hap_10 and Hap_11) in G. soja population was obviously decreased in G. max population, which might indicate that some new haplotypes were formed and some old haplotypes were lost during the G. max domesticated from G. soja.

[The mechanism of root hair development and molecular regulation in plants].

The formation of the root epidermis in Arabidopsis thaliana provides a simple model to study mechanisms underlying patterning in plants. Root hair increases the root surface area and effectively increases the root diameter, so root hair is thought to aid plants in nutrient uptake, anchorage and microbe interactions. The determination of root hair development has two types, lateral inhibition with feedback and position-dependent pattern of cell differentiation. The initiation and development of root hair in Arabidopsis provide a simple and efficacious model for the study of cell fate determination in plants. Molecular genetic studies identify a suite of putative transcription factors which regulate the epidermal cell pattern. The homeodomain protein GLABRA2 (GL2), R2R3 MYB-type transcription factor WEREWOLF (WER) and WD-repeat protein TRANSPARENTT TESTA GLABRA (TTG) are required for specification of non-hair cell type. The CAPRICE (CPC) and TRYPTICHON (TRY) are involved in specifying the hair cell fate.

[Genetic diversity and recombination of soybean cultivar Suinong 14 and its pedigree].

In this study, 14 agronomic traits and 139 SSR loci, distributed on 20 linkage groups of soybean (Glycine max L.) cultivar Suinong 14 and its pedigree were analyzed to explain the genetic diversity and recombination of Suinong 14 and to provide useful information for breeding. The cluster analysis based on SSR makers agreed with the pedigree information. The Shannon-Weaver index of each SSR locus ranged from 0 to 1.677. The average genetic similarity coefficient among cultivars was 0.6380, ranged from 0.538 to 0.799. At least three SSR loci were needed to discriminate Suinong 14 from its pedigree, for example a combination of Satt543, Sat_130 and Satt218. These loci have more alleles. No significant difference was observed between the end portion and the mid-portion within a linkage group, which indicates that the distribution of recombination occurred randomly in each linkage group. No polymorphism was detected within 39 of 139 SSR loci between Suinong 14 and its 8 parents. It implys their importance during cultivar improvement. Satt168, a marker on LGB2, was the only locus transmitted from Zihua 4 to Suinong 14, which indicates that the genetic constitute of Suinong 14 is greatly changed compared with Zihua 4 through five generations of recombination.