Genetic Diversity in Bradyrhizobium japonicum Serogroup 123 and Its Relation to Genotype-Specific Nodulation of Soybean

The genetic diversity among 20 field isolates of Bradyrhizobium japonicum serogroup 123 was examined by using restriction endonuclease digestions, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell proteins, Southern hybridization analysis of nif and nod genes, and intrinsic antibiotic resistance profiles. All of the isolates were previously separated into three broad nodulation classes (low, medium, and high) based on their ability to form symbioses with specific soybean genotypes. Results of our studies indicate that there is a relationship between these three genotype-specific nodulation classes and groupings that have been made based on genomic DNA digestion patterns, sodium dodecyl sulfate-protein profiles, and Southern hybridizations to a nifHD gene probe. Intrinsic antibiotic resistance profiles and nodAB gene hybridizations were not useful in determining interrelationships between isolates and nodulation classes. Southern hybridizations revealed that two of the isolates had reiterated nod genes; however, there was no correlation between the presence of extra nodAB genes and the nodulation classes or symbiotic performance on permissive soybean genotypes. Hybridizations with the nif gene probe indicated that there is a relationship among serogroup, nodulation class, and the physical organization of the genome.     

Nodulation and Competition for Nodulation of Selected Soybean Genotypes among Bradyrhizobium japonicum Serogroup 123 Isolates

Twenty recently obtained field isolates of Bradyrhizobium japonicum serogroup 123 were tested for their nodule mass production on the standard commercial soybean (Glycine max (L.) Merr. cv. Williams) and on two soybean plant introduction (PI) genotypes previously determined to restrict nodulation by strain USDA 123. Four of the field isolates showed similar restricted nodulation on the two genotypes, while all 20 isolates produced a normal amount of nodules on G. max cv. Williams. Serological analyses with adsorbed fluorescent antibodies showed that members of the 123 serotype ranked low in nodulation of the two PIs, in contrast to members of serotypes 127 and 129. Competition studies on the PIs indicated that isolates which were restricted were not competitive for nodule occupancy against strain USDA 110. However, unrestricted isolates of serogroup 123 were very competitive against USDA 110. On G. max cv. Williams, all serogroup 123 isolates tested were very competitive against USDA 110.     

Discovery of SNPs in soybean genotypes frequently used as the parents of mapping populations in the United States and Korea

Single nucleotide polymorphisms (SNPs) including insertion/deletions (indels) serve as useful and informative genetic markers. The availability of high-throughput and inexpensive SNP typing systems has increased interest in the development of SNP markers. After fragments of genes were amplified with primers derived from 110 soybean GenBank ESTs, sequencing data of PCR products from 15 soybean genotypes from Korea and the United States were analyzed by SeqScape software to find SNPs. Among 35 gene fragments with at least one SNP among the 15 genotypes, SNPs occurred at a frequency of 1 per 2,038 bp in 16,302 bp of coding sequence and 1 per 191 bp in 16,960 bp of noncoding regions. This corresponds to a nucleotide diversity (theta) of 0.00017 and 0.00186, respectively. Of the 97 SNPs discovered, 78 or 80.4% were present in the six North American soybean mapping parents. The addition of "Hwaeomputkong," which originated from Japan, increased the number to 92, or 94.8% of the total number of SNPs present among the 15 genotypes. Thus, Hwaeomputkong and the six North American mapping parents provide a diverse set of soybean genotypes that can be successfully used for SNP discovery in coding DNA and closely associated introns and untranslated regions.     

Expression and genome organization of resistance gene analogs in soybean.

Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes.     

The use of microsatellite DNA markers for soybean genotype identification

Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT) _n /(TA) _n and (ATT) _n /(TAA) _n that are composed of tandemly repeated 2–5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, n, results in PCR product length differences. The SSR alleles present at three (AT) _n /(TA) _n and four (ATT) _n /(TAA) _n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.     

Positive role of nodulation on the establishment ofRhizobium japonicum in subsequent crops of soybean

The influence of soybean nodulation on the establishment ofRhizobium japonicum inRhizobium-free soil was examined. Seeds of nodulating (Rj ₁) and nonnodulating (rj ₁) isolines of soybeans and four other crop species (cowpeas, mungbeans, corn, and alfalfa) were grown in field plots that were inoculated with a genetically marked strain ofRhizobium (strain I-110 ARS) and the following year nodulating soybeans were grown in these plots and were inoculated with a different genetically marked subline of the same strain (strain I-110 FN). The proportion of nodules containing strain I-110 ARS relative to strain I-110 FN was determined and interpreted as reflecting the relative numbers of the two genetically marked sublines in the soil. The results clearly demonstrate that nodulation with the specific host plant (soybeans) has a significant positive role in the establishment ofRhizobium inRhizobium-free soil and suggests that alfalfa plants diminish the establishment of soybean rhizobia in soil.     

Genetics of nitrogen metabolism and physiological/biochemical selection for increased grain crop productivity

Summary It is necessary to increase protein productivity of grain crops to meet present and future world protein requirements. Conventional plant breeding methodology has been to select genotypes with enhanced yield or grain protein concentration. In addition to this determination of end product, we suggest measurements of a number of physiological and biochemical processes of nitrogen (N) metabolism which precede plant maturity as selection criteria for enhanced N metabolism and grain crop productivity. The measurement across the growing season of genotypic variation in components of N metabolism would constitute a physiological/biochemical selection program to be incorporated with the determination of harvestable end product. A properly designed physiological/biochemical selection program would integrate the effects of plant genotype, environment, and their interactions allowing identification of the factors limiting productivity of particular genotypes, and would also estimate end product. Our review of literature pertinent to whole plant N metabolism suggests that such a selection program initially include NO ₃ ^- uptake, N₂ fixation, N accumulation, N remobilization, seed protein synthesis, and Nitrogen Harvest Index.Supported by USDA, Nitrogen Fixation and Soybean Genetics Laboratory, Beltsville, Md., and the Agronomy Department, University of Maryland, College Park, Md., under Cooperative Agreement 58-32U4-3-370, Scientific Article No. A-3400, Contribution No. 6473 of the Maryland Agricultural Experiment Station, Department of Agronomy, College Park, MD 20742, USA     

Genomic consequences of selection and genome-wide association mapping in soybean

Background

Crop improvement always involves selection of specific alleles at genes controlling traits of agronomic importance, likely resulting in detectable signatures of selection within the genome of modern soybean (Glycine max L. Merr.). The identification of these signatures of selection is meaningful from the perspective of evolutionary biology and for uncovering the genetic architecture of agronomic traits.

Results

To this end, two populations of soybean, consisting of 342 landraces and 1062 improved lines, were genotyped with the SoySNP50K Illumina BeadChip containing 52,041 single nucleotide polymorphisms (SNPs), and systematically phenotyped for 9 agronomic traits. A cross-population composite likelihood ratio (XP-CLR) method was used to screen the signals of selective sweeps. A total of 125 candidate selection regions were identified, many of which harbored genes potentially involved in crop improvement. To further investigate whether these candidate regions were in fact enriched for genes affected by selection, genome-wide association studies (GWAS) were conducted on 7 selection traits targeted in soybean breeding (grain yield, plant height, lodging, maturity date, seed coat color, seed protein and oil content) and 2 non-selection traits (pubescence and flower color). Major genomic regions associated with selection traits overlapped with candidate selection regions, whereas no overlap of this kind occurred for the non-selection traits, suggesting that the selection sweeps identified are associated with traits of agronomic importance. Multiple novel loci and refined map locations of known loci related to these traits were also identified.

Conclusions

These findings illustrate that comparative genomic analyses, especially when combined with GWAS, are a promising approach to dissect the genetic architecture of complex traits.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1872-y) contains supplementary material, which is available to authorized users.