Mutational analysis of the major soybean UreF paralogue involved in urease activation

The soybean genome duplicated ∼14 and 45 million years ago and has many paralogous genes, including those in urease activation (emplacement of Ni and CO2 in the active site). Activation requires the UreD and UreF proteins, each encoded by two paralogues. UreG, a third essential activation protein, is encoded by the single-copy Eu3, and eu3 mutants lack activity of both urease isozymes. eu2 has the same urease-negative phenotype, consistent with Eu2 being a single-copy gene, possibly encoding a Ni carrier. Unexpectedly, two eu2 alleles co-segregated with missense mutations in the chromosome 2 UreF paralogue (Ch02UreF), suggesting lack of expression/function of Ch14UreF. However, Ch02UreF and Ch14UreF transcripts accumulate at the same level. Further, it had been shown that expression of the Ch14UreF ORF complemented a fungal ureF mutant. A third, nonsense (Q2*) allelic mutant, eu2-c, exhibited 5- to 10-fold more residual urease activity than missense eu2-a or eu2-b, though eu2-c should lack all Ch02UreF protein. It is hypothesized that low-level activation by Ch14UreF is ‘spoiled’ by the altered missense Ch02UreF proteins (‘epistatic dominant-negative’). In agreement with active ‘spoiling’ by eu2-b-encoded Ch02UreF (G31D), eu2-b/eu2-c heterozygotes had less than half the urease activity of eu2-c/eu2-c siblings. Ch02UreF (G31D) could spoil activation by Chr14UreF because of higher affinity for the activation complex, or because Ch02UreF (G31D) is more abundant than Ch14UreF. Here, the latter is favoured, consistent with a reported in-frame AUG in the 5' leader of Chr14UreF transcript. Translational inhibition could represent a form of ‘functional divergence’ of duplicated genes

Phytophthora Root Rot Resistance in Soybean E00003

Phytophthora root rot (PRR) is a devastating disease in soybean [Glycine max (L.) Merr.] production. Michigan elite soybean E00003 is resistant to Phytophthora sojae and has been used as a resistance source in breeding. Genetic control of PRR resistance in this source is unknown. To facilitate marker-assisted selection (MAS), the PRR resistance loci in E00003 and their map locations need to be determined. In this study, a genetic mapping approach was used to identify major PRR -resistant loci in E00003. The mapping population consists of 240 F4–derived lines developed by crossing E00003 with the P. sojae susceptible line PI 567543C. In 2009 and 2010, the mapping population was evaluated in the greenhouse for PRR resistance against P. sojae races 1, 4, and 7, using modified rice (Oryza sativa L.) grain inoculation method. The population was genotyped with seven simple sequence repeat (SSR) and three single nucleotide polymorphism (SNP) markers derived from bulk segregant analysis. The heritability of resistance in the population ranged from 83 to 94%. A major locus, contributing 50 to 76% of the phenotypic variation, was mapped within a 3 cM interval in the Rps1 region. The interval was further saturated with more BARCSOY SSRs and SNPs with TaqMan assays. Two SSRs and three SNPs within the Rps1k gene were highly associated with PRR resistance in the mapping population. The major resistance gene in E00003 is either allelic or tightly linked to Rps1k.The molecular markers located in the Rps1k gene can be used to improve MAS for PRR resistance

High-throughput SNP discovery through deep resequencing of a reduced representation library to anchor and orient scaffolds in the soybean whole genome sequence

Background: The Soybean Consensus Map 4.0 facilitated the anchoring of 95.6% of the soybean whole genome sequence developed by the Joint Genome Institute, Department of Energy, but its marker density was only sufficient to properly orient 66% of the sequence scaffolds. The discovery and genetic mapping of more single nucleotide polymorphism (SNP) markers were needed to anchor and orient the remaining genome sequence. To that end, next generation sequencing and high-throughput genotyping were combined to obtain a much higher resolution genetic map that could be used to anchor and orient most of the remaining sequence and to help validate the integrity of the existing scaffold builds. Results: A total of 7,108 to 25,047 predicted SNPs were discovered using a reduced representation library that was subsequently sequenced by the Illumina sequence-by-synthesis method on the clonal single molecule array platform. Using multiple SNP prediction methods, the validation rate of these SNPs ranged from 79% to 92.5%. A high resolution genetic map using 444 recombinant inbred lines was created with 1,790 SNP markers. Of the 1,790 mapped SNP markers, 1,240 markers had been selectively chosen to target existing un-anchored or un-oriented sequence scaffolds, thereby increasing the amount of anchored sequence to 97%. Conclusion: We have demonstrated how next generation sequencing was combined with high-throughput SNP detection assays to quickly discover large numbers of SNPs. Those SNPs were then used to create a high resolution genetic map that assisted in the assembly of scaffolds from the 8× whole genome shotgun sequences into pseudomolecules corresponding to chromosomes of the organism

Genomic regions that underlie soybean seed isoflavone content

Soy products contain isoflavones (genistein, daidzein, and glycitein)that display biological effects when ingested by humans and animals, these effects are species, dose and age dependent. Therefore, the content and quality of isoflavones in soybeans is a key to their biological effect. Our objective was to identify loci that underlie isoflavone content in soybean seeds. The study involved 100 recombinant inbred lines (RIL)fr om the cross of ‘Essex’ by ‘Forrest,’ two cultivars that contrast for isoflavone content. Isoflavone content of seeds fromeach RIL was determined by high performance liquid chromatography (HPLC). The distribution of isoflavone content was continuous and unimodal. The heritability estimates on a line mean basis were 79% for daidzein, 22% for genistein, and 88% for glycitein. Isoflavone content of soybean seeds was compared against 150 polymorphic DNA markers in a one-way analysis of variance. Four genomic regions were found to be significantly associated with the isoflavone content of soybean seeds across both locations and years. Molecular linkage group B1 contained a major QTL underlying glycitein content (P = 0.0001,R2 = 50.2%), linkage group N contained a QTL for glycitein (P = 0.0033,R2 = 11.1%)and a QTL for daidzein (P = 0.0023,R2 = 10.3%) and linkage group A1 contained a QTL for daidzein (P = 0.0081,R2 = 9.6%). Selection for these chromosomal regions in a marker assisted selection program will allow for the manipulation of amounts and profiles of isoflavones (genistein, daidzein, and glycitein)c ontent of soybean seeds. In addition, tightly linked markers can be used in map based cloning of genes associated with isoflavone content

QTL for seed protein and amino acids in the Benning × Danbaekkong soybean population

Soybean, rather than nitrogen-containing forages, is the primary source of quality protein in feed formulations for domestic swine, poultry, and dairy industries. As a sole dietary source of protein, soybean is deficient in the amino acids lysine (Lys), threonine (Thr), methionine (Met), and cysteine (Cys). Increasing these amino acids would benefit the feed industry. The objective of the present study was to identify quantitative trait loci (QTL) associated with crude protein (cp) and amino acids in the ‘Benning’ × ‘Danbaekkong’ population. The population was grown in five southern USA environments. Amino acid concentrations as a fraction of cp (Lys/cp, Thr/cp, Met/cp, Cys/cp, and Met + Cys/cp) were determined by near-infrared reflectance spectroscopy. Four QTL associated with the variation in crude protein were detected on chromosomes (Chr) 14, 15, 17, and 20, of which, a QTL on Chr 20 explained 55 % of the phenotypic variation. In the same chromosomal region, QTL for Lys/cp, Thr/cp, Met/cp, Cys/cp and Met + Cys/cp were detected. At these QTL, the Danbaekkong allele resulted in reduced levels of these amino acids and increased protein concentration. Two additional QTL for Lys/cp were detected on Chr 08 and 20, and three QTL for Thr/cp on Chr 01, 09, and 17. Three QTL were identified on Chr 06, 09 and 10 for Met/cp, and one QTL was found for Cys/cp on Chr 10. The study provides information concerning the relationship between crude protein and levels of essential amino acids and may allow for the improvement of these traits in soybean using marker-assisted selection

Genetic Characterization of the Soybean Nested Association Mapping Population

A set of nested association mapping (NAM) families was developed by crossing 40 diverse soybean [Glycine max (L.) Merr.] genotypes to the common cultivar. The 41 parents were deeply sequenced for SNP discovery. Based on the polymorphism of the single-nucleotide polymorphisms (SNPs) and other selection criteria, a set of SNPs was selected to be included in the SoyNAM6K BeadChip for genotyping the parents and 5600 RILs from the 40 families. Analysis of the SNP profiles of the RILs showed a low average recombination rate. We constructed genetic linkage maps for each family and a composite linkage map based on recombinant inbred lines (RILs) across the families and identified and annotated 525,772 high confidence SNPs that were used to impute the SNP alleles in the RILs. The segregation distortion in most families significantly favored the alleles from the female parent, and there was no significant difference of residual heterozygosity in the euchromatic vs. heterochromatic regions. The genotypic datasets for the RILs and parents are publicly available and are anticipated to be useful to map quantitative trait loci (QTL) controlling important traits in soybean

SNP assay to detect the ‘Hyuuga’ red-brown lesion resistance gene for Asian soybean rust

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi Syd., has the potential to become a serious threat to soybean, Glycine max L. Merr., production in the USA. A novel rust resistance gene, Rpp?(Hyuuga), from the Japanese soybean cultivar Hyuuga has been identified and mapped to soybean chromosome 6 (Gm06). Our objectives were to fine-map the Rpp?(Hyuuga) gene and develop a high-throughput single nucleotide polymorphism (SNP) assay to detect this ASR resistance gene. The integration of recombination events from two different soybean populations and the ASR reaction data indicates that the Rpp?(Hyuuga) locus is located in a region of approximately 371 kb between STS70887 and STS70923 on chromosome Gm06. A set of 32 ancestral genotypes which is predicted to contain 95% of the alleles present in current elite North American breeding populations and the sources of the previously reported ASR resistance genes (Rpp1, Rpp2, Rpp3, Rpp4, Rpp5, and rpp5) were genotyped with five SNP markers. We developed a SimpleProbe assay based on melting curve analysis for SNP06-44058 which is tighly linked to the Rpp?(Hyuuga) gene. This SNP assay can differentiate plants/lines that are homozygous/homogeneous or heterozygous/heterogeneous for the resistant and susceptible alleles at the Rpp?(Hyuuga) locus

Defining the Transcriptome Assembly and Its Use for Genome Dynamics and Transcriptome Profiling Studies in Pigeonpea (Cajanus cajan L.)

This study reports generation of large-scale genomic resources for pigeonpea, a so-called ‘orphan crop species’ of the semi-arid tropic regions. FLX/454 sequencing carried out on a normalized cDNA pool prepared from 31 tissues produced 494 353 short transcript reads (STRs). Cluster analysis of these STRs, together with 10 817 Sanger ESTs, resulted in a pigeonpea trancriptome assembly (CcTA) comprising of 127 754 tentative unique sequences (TUSs). Functional analysis of these TUSs highlights several active pathways and processes in the sampled tissues. Comparison of the CcTA with the soybean genome showed similarity to 10 857 and 16 367 soybean gene models (depending on alignment methods). Additionally, Illumina 1G sequencing was performed on Fusarium wilt (FW)- and sterility mosaic disease (SMD)-challenged root tissues of 10 resistant and susceptible genotypes. More than 160 million sequence tags were used to identify FW- and SMD-responsive genes. Sequence analysis of CcTA and the Illumina tags identified a large new set of markers for use in genetics and breeding, including 8137 simple sequence repeats, 12 141 single-nucleotide polymorphisms and 5845 intron-spanning regions. Genomic resources developed in this study should be useful for basic and applied research, not only for pigeonpea improvement but also for other related, agronomically important legumes

Genetic Architecture of Soybean Yield and Agronomic Traits

Soybean is the world’s leading source of vegetable protein and demand for its seed continues to grow. Breeders have successfully increased soybean yield, but the genetic architecture of yield and key agronomic traits is poorly understood. We developed a 40-mating soybean nested association mapping (NAM) population of 5,600 inbred lines that were characterized by single nucleotide polymorphism (SNP) markers and six agronomic traits in field trials in 22 environments. Analysis of the yield, agronomic, and SNP data revealed 23 significant marker-trait associations for yield, 19 for maturity, 15 for plant height, 17 for plant lodging, and 29 for seed mass. A higher frequency of estimated positive yield alleles was evident from elite founder parents than from exotic founders, although unique desirable alleles from the exotic group were identified, demonstrating the value of expanding the genetic base of US soybean breeding