Genetic variation and genetic complexity of nodule occupancy in soybean inoculated with USDA110 and USDA123 rhizobium strains.

BACKGROUND: Symbiotic nitrogen fixation differs among Bradyrhizobium japonicum strains. Soybean inoculated with USDA123 has a lower yield than strains known to have high nitrogen fixation efficiency, such as USDA110. In the main soybean-producing area in the Midwest of the United States, USDA123 has a high nodule incidence in field-grown soybean and is competitive but inefficient in nitrogen fixation. In this study, a high-throughput system was developed to characterize nodule number among 1,321 Glycine max and 69 Glycine soja accessions single inoculated with USDA110 and USDA123. RESULTS: Seventy-three G. max accessions with significantly different nodule number of USDA110 and USDA123 were identified. After double inoculating 35 of the 73 accessions, it was observed that PI189939, PI317335, PI324187B, PI548461, PI562373, and PI628961 were occupied by USDA110 and double-strain nodules but not by USDA123 nodules alone. PI567624 was only occupied by USDA110 nodules, and PI507429 restricted all strains. Analysis showed that 35 loci were associated with nodule number in G. max when inoculated with strain USDA110 and 35 loci with USDA123. Twenty-three loci were identified in G. soja when inoculated with strain USDA110 and 34 with USDA123. Only four loci were common across two treatments, and each locus could only explain 0.8 to 1.5% of phenotypic variation. CONCLUSIONS: High-throughput phenotyping systems to characterize nodule number and occupancy were developed, and soybean germplasm restricting rhizobium strain USDA123 but preferring USDA110 was identified. The larger number of minor effects and a small few common loci controlling the nodule number indicated trait genetic complexity and strain-dependent nodulation restriction. The information from the present study will add to the development of cultivars that limit USDA123, thereby increasing nitrogen fixation efficiency and productivity.

Genotype imputation for soybean nested association mapping population to improve precision of QTL detection.

KEY MESSAGE: Software for high imputation accuracy in soybean was identified. Imputed dataset could significantly reduce the interval of genomic regions controlling traits, thus greatly improve the efficiency of candidate gene identification. Genotype imputation is a strategy to increase marker density of existing datasets without additional genotyping. We compared imputation performance of software BEAGLE 5.0, IMPUTE 5 and AlphaPlantImpute and tested software parameters that may help to improve imputation accuracy in soybean populations. Several factors including marker density, extent of linkage disequilibrium (LD), minor allele frequency (MAF), etc., were examined for their effects on imputation accuracy across different software. Our results showed that AlphaPlantImpute had a higher imputation accuracy than BEAGLE 5.0 or IMPUTE 5 tested in each soybean family, especially if the study progeny were genotyped with an extremely low number of markers. LD extent, MAF and reference panel size were positively correlated with imputation accuracy, a minimum number of 50 markers per chromosome and MAF of SNPs > 0.2 in soybean line were required to avoid a significant loss of imputation accuracy. Using the software, we imputed 5176 soybean lines in the soybean nested mapping population (NAM) with high-density markers of the 40 parents. The dataset containing 423,419 markers for 5176 lines and 40 parents was deposited at the Soybase. The imputed NAM dataset was further examined for the improvement of mapping quantitative trait loci (QTL) controlling soybean seed protein content. Most of the QTL identified were at identical or at similar position based on initial and imputed datasets; however, QTL intervals were greatly narrowed. The resulting genotypic dataset of NAM population will facilitate QTL mapping of traits and downstream applications. The information will also help to improve genotyping imputation accuracy in self-pollinated crops.

Soybean BARCSoySNP6K: An assay for soybean genetics and breeding research.

The limited number of recombinant events in recombinant inbred lines suggests that for a biparental population with a limited number of recombinant inbred lines, it is unnecessary to genotype the lines with many markers. For genomic prediction and selection, previous studies have demonstrated that only 1000-2000 genome-wide common markers across all lines/accessions are needed to reach maximum efficiency of genomic prediction in populations. Evaluation of too many markers will not only increase the cost but also generate redundant information. We developed a soybean (Glycine max) assay, BARCSoySNP6K, containing 6000 markers, which were carefully chosen from the SoySNP50K assay based on their position in the soybean genome and haplotype block, polymorphism among accessions and genotyping quality. The assay includes 5000 single nucleotide polymorphisms (SNPs) from euchromatic and 1000 from heterochromatic regions. The percentage of SNPs with minor allele frequency >0.10 was 95% and 91% in the euchromatic and heterochromatic regions, respectively. Analysis of progeny from two large families genotyped with SoySNP50K versus BARCSoySNP6K showed that the position of the common markers and number of unique bins along linkage maps were consistent based on the SNPs genotyped with the two assays; however, the rate of redundant markers was dramatically reduced with the BARCSoySNP6K. The BARCSoySNP6K assay is proven as an excellent tool for detecting quantitative trait loci, genomic selection and assessing genetic relationships. The assay is commercialized by Illumina Inc. and being used by soybean breeders and geneticists and the list of SNPs in the assay is an ideal resource for SNP genotyping by targeted amplicon sequencing.

Microsatellite marker development by partial sequencing of the sour passion fruit genome (Passiflora edulis Sims).

BACKGROUND: The Passiflora genus comprises hundreds of wild and cultivated species of passion fruit used for food, industrial, ornamental and medicinal purposes. Efforts to develop genomic tools for genetic analysis of P. edulis, the most important commercial Passiflora species, are still incipient. In spite of many recognized applications of microsatellite markers in genetics and breeding, their availability for passion fruit research remains restricted. Microsatellite markers in P. edulis are usually limited in number, show reduced polymorphism, and are mostly based on compound or imperfect repeats. Furthermore, they are confined to only a few Passiflora species. We describe the use of NGS technology to partially assemble the P. edulis genome in order to develop hundreds of new microsatellite markers. RESULTS: A total of 14.11 Gbp of Illumina paired-end sequence reads were analyzed to detect simple sequence repeat sites in the sour passion fruit genome. A sample of 1300 contigs containing perfect repeat microsatellite sequences was selected for PCR primer development. Panels of di- and tri-nucleotide repeat markers were then tested in P. edulis germplasm accessions for validation. DNA polymorphism was detected in 74% of the markers (PIC = 0.16 to 0.77; number of alleles/locus = 2 to 7). A core panel of highly polymorphic markers (PIC = 0.46 to 0.77) was used to cross-amplify PCR products in 79 species of Passiflora (including P. edulis), belonging to four subgenera (Astrophea, Decaloba, Distephana and Passiflora). Approximately 71% of the marker/species combinations resulted in positive amplicons in all species tested. DNA polymorphism was detected in germplasm accessions of six closely related Passiflora species (P. edulis, P. alata, P. maliformis, P. nitida, P. quadrangularis and P. setacea) and the data used for accession discrimination and species assignment. CONCLUSION: A database of P. edulis DNA sequences obtained by NGS technology was examined to identify microsatellite repeats in the sour passion fruit genome. Markers were submitted to evaluation using accessions of cultivated and wild Passiflora species. The new microsatellite markers detected high levels of DNA polymorphism in sour passion fruit and can potentially be used in genetic analysis of P. edulis and other Passiflora species.

Genetic Diversity and Phylogenetic Relationships of Annual and Perennial Glycine Species.

We have estimated the average genetic diversity of two Glycine annual and six perennial species based upon 76 orthologous gene sets and performed phylogenetic analysis, divergence analysis and tests for departure from neutrality of the eight species using 52 orthologous gene sets. In addition, 367 orthologous gene sets were used to estimate the relationships of 11 G. canescens accessions. Among the perennials, G. canescens showed the highest nucleotide diversity. The other perennials, except for G. tomentella, had higher nucleotide diversity than the two annuals. Phylogenetic analysis of the Glycine showed a similar genome grouping with the previous report except for G. cyrtoloba and G. stenophita which formed a sister clade in the study. Divergence analysis supported the phylogenetic relationships that G. falcata was the most divergent from G. max, followed by G. cyrtoloba, G. syndetika, G. tomentella D3, G. stenophita and G. canescens Most genic sequences were homogeneous in the levels of polymorphism and divergence between G. max and other Glycine species based on the HKA test, thus, Glycine perennials may have experienced a very similar evolution as inferred by trans-specific mutation analysis. The greater genetic diversity of most perennial Glycine species and their origins from the warmer and drier climates of Australia suggests the perennials maybe a potential source of heat and drought resistance that will be of value in the face of climate change.