Articles

Metabolite Fingerprinting in Transgenic Nicotiana tabacum Altered by the Escherichia coli Glutamate Dehydrogenase Gene

R. Mungur et al. — Mon, 19 Apr 2010 07:20:40 PDT

With about 200 000 phytochemicals in existence, identifying those of biomedical significance is a mammoth task. In the postgenomic era, relating metabolite fingerprints, abundances, and profiles to genotype is also a large task. Ion analysis using Fourier transformed ion cyclotron resonance mass spectrometry (FT-ICR-MS) may provide a high-throughput approach to measure genotype dependency of the inferred metabolome if reproducible techniques can be established. Ion profile inferred metabolite fingerprints are coproducts. We used FT-ICR-MS-derived ion analysis to examine gdhA (glutamate dehydrogenase (GDH; EC 1.4.1.1)) transgenic Nicotiana tabacum (tobacco) carrying out altered glutamate, amino acid, and carbon metabolisms, that fundamentally alter plant productivity. Cause and effect between gdhA expression, glutamate metabolism, and plant phenotypes was analyzed by 13NH4+ labeling of amino acid fractions, and by FT-ICR-MS analysis of metabolites. The gdhA transgenic plants increased 13N labeling of glutamate and glutamine significantly. FT-ICR-MS detected 2 012 ions reproducible in 2 to 4 ionization protocols. There were 283 ions in roots and 98 ions in leaves that appeared to significantly change abundance due to the measured GDH activity. About 58% percent of ions could not be used to infer a corresponding metabolite. From the 42% of ions that inferred known metabolites we found that certain amino acids, organic acids, and sugars increased and some fatty acids decreased. The transgene caused increased ammonium assimilation and detectable ion variation. Thirty-two compounds with biomedical significance were altered in abundance by GDH including 9 known carcinogens and 14 potential drugs. Therefore, the GDH transgene may lead to new uses for crops like tobacco.

Separate loci underlie resistance to root infection and leaf scorch during soybean sudden death syndrome

Samreen Kazi et al. — Mon, 22 Mar 2010 07:40:06 PDT

Soybean [Glycine max (L.) Merr.] cultivars show differences in their resistance to both the leaf scorch and root rot of sudden death syndrome (SDS). The syndrome is caused by root colonization by Fusarium virguliforme (ex. F. solani f. sp. glycines). Root susceptibility combined with reduced leaf scorch resistance has been associated with resistance to Heterodera glycines HG Type 1.3.6.7 (race 14) of the soybean cyst nematode (SCN). In contrast, the rhg1 locus underlying resistance to Hg Type 0 was found clustered with three loci for resistance to SDS leaf scorch and one for root infection. The aims of this study were to compare the inheritance of resistance to leaf scorch and root infection in a population that segregated for resistance to SCN and to identify the underlying quantitative trait loci (QTL). “Hartwig”, a cultivar partially resistant to SDS leaf scorch, F. virguliforme root infection and SCN HG Type 1.3.6.7 was crossed with the partially susceptible cultivar “Flyer”. Ninety-two F5-derived recombinant inbred lines and 144 markers were used for map development. Four QTL found in earlier studies were confirmed. One contributed resistance to leaf scorch on linkage group (LG) C2 (Satt277; P = 0.004, R 2 = 15%). Two on LG G underlay root infection at R8 (Satt038; P = 0.0001 R 2 = 28.1%; Satt115; P = 0.003, R 2 = 12.9%). The marker Satt038 was linked to rhg1 underlying resistance to SCN Hg Type 0. The fourth QTL was on LG D2 underlying resistance to root infection at R6 (Satt574; P = 0.001, R 2 = 10%). That QTL was in an interval previously associated with resistance to both SDS leaf scorch and SCN Hg Type 1.3.6.7. The QTL showed repulsion linkage with resistance to SCN that may explain the relative susceptibility to SDS of some SCN resistant cultivars. One additional QTL was discovered on LG G underlying resistance to SDS leaf scorch measured by disease index (Satt130; P = 0.003, R 2 = 13%). The loci and markers will provide tagged alleles with which to improve the breeding of cultivars combining resistances to SDS leaf scorch, root infection and SCN HG Type 1.3.6.7.

Iso-lines and inbred-lines confirmed loci that underlie resistance from cultivar ‘Hartwig’ to three soybean cyst nematode populations

Samreen Kazi et al. — Mon, 22 Mar 2010 07:20:05 PDT

Soybean [Glycine max (L.) Merr.] cultivars varied in their resistance to different populations of the soybean cyst nematode (SCN), Heterodera glycines, called HG Types. The rhg1 locus on linkage group G was necessary for resistance to all HG types. However, the loci for resistance to H. glycines HG Type 1.3- (race 14) and HG Type 1.2.5- (race 2) of the soybean cyst nematode have varied in their reported locations. The aims were to compare the inheritance of resistance to three nematode HG Types in a population segregating for resistance to SCN and to identify the underlying quantitative trait loci (QTL). ‘Hartwig’, a soybean cultivar resistant to most SCN HG Types, was crossed with the susceptible cultivar ‘Flyer’. A total of 92 F5-derived recombinant inbred lines (RILs; or inbred lines) and 144 molecular markers were used for map development. The rhg1 associated QTL found in earlier studies were confirmed and shown to underlie resistance to all three HG Types in RILs (Satt309; HG Type 0, P = 0.0001 R 2 = 22%; Satt275; HG Type 1.3, P = 0.001, R 2 = 14%) and near isogeneic lines (NILs; or iso-lines; Satt309; HG Type 1.2.5-, P = 0.001 R 2 = 24%). A new QTL underlying resistance to HG Type 1.2.5- was detected on LG D2 (Satt574; P = 0.001, R 2 = 11%) among 14 RILs resistant to the other HG types. The locus was confirmed in a small NIL population consisting of 60 plants of ten genotypes (P = 0.04). This QTL (cqSCN-005) is located in an interval previously associated with resistance to both SDS leaf scorch from ‘Pyramid’ and ‘Ripley’ (cqSDS-001) and SCN HG Type 1.3- from Hartwig and Pyramid. The QTL detected will allow marker assisted selection for multigenic resistance to complex nematode populations in combination with sudden death syndrome resistance (SDS) and other agronomic traits.

A High-Resolution Melting Approach for Analyzing Allelic Expression Dynamics

Jiazheng Yuan et al. — Thu, 11 Mar 2010 14:19:42 PST

Single nucleotide polymorphisms (SNPs) are single base pair mutations that provide new approaches to studies of allele transcript abundances. High-resolution DNA melting curve (HRM) analysis using a LightScanner (Hi-Res Melting system with Idaho's LC Green) provides post-PCR detection of mutations and SNPs in genomic DNA. This study investigated whether the HRM analysis can distinguish alleles among potato (Solanum tuberosum) transcript abundances. Transcript properties of genes encoding seven carbohydrate metabolism enzymes/proteins in various tissues and cold storage durations were studied. The HRM assay measured differential expression of alleles between different organs, between different storage treatments and stages of tubers from the same variety, and between different varieties with the same treatment. The RT-PCR amplicons were directly sequenced to assist the interpretation of HRM data. The cDNA HRM curves correlated well with the nucleotide polymorphisms of the cDNA sequences and the transcript abundance of alleles and therefore can serve as functional allele activity (FAA) markers. By combining the allelic specificity of HRM with simple PCR design, this technology can be applied to rapidly determine the most active allele of a gene among the cells analyzed.

Multigeneic QTL: The Laccase Encoded within the Soybean Rfs2/rhg1 Locus Inferred to Underlie Part of the Dual Resistance to Cyst Nematode and Sudden Death Syndrome

M Javed Iqbal et al. — Thu, 11 Mar 2010 14:19:41 PST

Multigeneic QTL present significant problems to analysis. Resistance to soybean (Glycine max (L) Merr.) sudden death syndrome (SDS) caused by Fusarium virguliforme was partly underlain by QRfs2 that was clustered with, or pleiotropic to, the multigeneic rhg1 locus providing resistance to soybean cyst nematode (SCN; Heterodera glycines ). A group of five genes were found between the two markers that delimited the Rfs2/rhg1 locus. One of the five genes was predicted to encode an unusual diphenol oxidase (laccase; EC 1.10.3.2). The aim of this study was to characterize this member of the soybean laccase gene-family and explore its involvement in SDS resistance. A genomic clone and a full length cDNA was isolated from resistant cultivar 'Forrest' that were different among susceptible cultivars 'Asgrow 3244' and 'Williams 82' at four residues R/H168, I/M271, R/H330, E/K470. Additional differences were found in six of the seven introns and the promoter region. Transcript abundance (TA) among genotypes that varied for resistance to SDS or SCN did not differ significantly. Therefore the protein activity was inferred to underlie resistance. Protein expressed in yeast pYES2/NTB had weak enzyme activity with common substrates but good activity with root phenolics. The Forrest isoform may underlie both QRfs 2 and rhg1

Antisense Phenotypes Reveal a Functional Expression of OsARF1, an Auxin Response Factor, in Transgenic Rice

Kotb A. Attia et al. — Thu, 11 Mar 2010 14:19:40 PST

OsARF1 is the first full-length member of auxin response factor (ARF) gene family to be cloned from monocot plant. Using quantitative RT-PCR this study found that, the transcript abundance of OsARF1 was significantly higher in embryonic tissues than in vegetative tissues. To investigate the effect of OsARF1 on the phenotype of rice, a cDNA fragment of OsARF1 was inserted in inverse orientation to the 35S promoter in vector pBin438 to produce an antisense (AS) construction. The AS-OsARF1 construct was transferred into rice (Oryza sativa L. japonica ) calli via Agrobacterium tumefaciens -mediated transformation. Molecular analysis of transgenic plants showed that the functional expression of OsARF1 was inhibited at mRNA level efficiently. The AS-OsARF1 plants showed extremely low growth, poor vigor, short curled leaves and tillered but were sterile. Therefore, the OsARF1 was shown to be essential for growth in vegetative organs and seed development.

Establishment of the Regeneration System for Vicia faba L.

Shimaa Bahgat et al. — Thu, 11 Mar 2010 14:19:39 PST

A reliable regeneration system for faba bean has been difficult to establish and therefore, the genetic improvement of Vicia faba L. was delayed. The paper describes a method of somatic embryo induction in callus of V. faba . Two Egyptian faba bean cultivars 'Giza 2' and '24 Hyto' were used. Callus was induced from epicotyls and shoot tips cultured on MS or Gamborg medium supplemented with 3% sucrose and 0.025% (w/v) for each of ascorbic and citric acid, 0.8% agar and different concentrations of 10 mg/l BAP, 0.5 mg/l of each NAA and 2,4-dichlorophenoxyacetic acid (M1) and 1 mg/l BAP and 0.5 mg/l NAA (M2) . The media with BAP, NAA and 2,4-D were optimal for embryogenic callus induction. Somatic embryos developed after transfer of the callus to 1/2 B5 medium with no plant growth regulators. There were various stages of somatic embryo development present including globular, heart-shaped, torpedo, and cotyledonary stages. Embryos developed into plantlets and plants were regenerated. RAPD analyses were performed to investigate the genetic stability of the regenerated plants obtained from different treatments and different explants. The cultivar Giza 2 exhibited more genetic stability than cultivar 24 Hyto. In conclusion, a regeneration system was established suitable for both gene transformation and the isolation of somaclonal mutants. The regeneration system will be used in order to improve the nutritional value of faba bean.

The Interactions of the Largest Subunit of RNA Polymerase II with Other Cellular Proteins: a Bioinformatic Approach

Abhijit Shukla et al. — Thu, 11 Mar 2010 14:19:38 PST

The function of a protein is governed by its interaction with other proteins inside a cell. Therefore, it is important to identify the interacting partners of a particular protein to decipher its function. The protein interaction networks are generally determined by bioinformatic as well as experimental methodologies such as yeast two hybrid, mass spectrometry, immunoprecipitation, and fluorescence resonance energy transfer assays. Here, we have analyzed bioinformatically the interactions of Rpb1p (the largest subunit of RNA Polymerase II) with other proteins in yeast, using Cytoscape software and Biogrid/Biomart database. We find that Rpb1p interacts with a large number of proteins involved in mRNA synthesis, processing, export, and other cellular processes. These results validate the application of such bioinformatic approach to determine the interactome for other cellular proteins.

Locus Interactions Underlie Seed Yield In Soybeans Resistant to Heterodera glycines

U B. Karangula et al. — Thu, 11 Mar 2010 14:19:37 PST

In soybean (Glycine max L. Merr.) combining resistance to cyst nematode (SCN; Heterodera glycines I.) with high seed yieldremains problematic. Molecular markers linked to quantitative trait loci (QTL) have not provided a solution. Sets of markers describing a collection of favorable alleles (linkats) may assist plant breeders seeking to combine both traits. The objective of this analysis was to identify linkats in genomic regions underlying seed yield and root SCN resistance QTL. Used were groups of cultivars selected from a single recombinant inbred (RIL) population derived from 'Essex' by 'Forrest' (ExF). The yield was measured at four locations. SCN resistance was determined in greenhouse assays. The mean seed yield was used to define 3 groups (each n = 30), high, medium and low. SCN resistance formed 2 groups (SCN resistant (n = 21) and SCN susceptible (n = 69)). Microsatellite markers (213) alleles were compared with seed yield and root SCN (Hetrodera glycines) resistance using mean analysis. The number, size and position of potential linkats were determined. Loci, genomic regions and linkats associated with seed yield were identified on linkage group (LG) K and with root resistance to SCN e on LG E, G, and D1b+W. A method to identify co-localized genomic regions is presented.

Different Responses of Two Genes Associated with Disease Resistance Loci in Maize (Zea mays L.) to 3-allyloxy-1,2-benzothiazole 1,1-dioxide

Jiazheng Yuan et al. — Thu, 11 Mar 2010 14:19:36 PST

Probenazole (3-allyloxy-1,2-benzothiazole 1,1-dioxide, PBZ) is a bactericide and fungicide that acts by inducing plant defense systems. It has been shown to induce the expression of NBS-LRR genes like RPR1 (rice probenazole-response gene) in rice (Oryza sativa L.) and systemic acquired resistance (SAR)-like disease resistance. Two maize (Zea mays L.) genes Zmnbslrr1 (a NBS-LRR gene, cloned from a disease resistance analog PIC11 based) and Zmgc1 , (a putative guanylyl cyclase-like gene) have both been associated with quantitative resistance loci (QTL) for resistance to Fusarium graminearum . PIC11 was associated with Fusarium stalk rot and ZmGC1 showed resistance to Gibberella ear rot caused by F. graminearum . The objectives of the current study here were to characterize the Zmnbslrr1 gene and to determine whether it and Zmgc1 respond to the inducer PBZ. The transcript abundance of Zmnbslrr1 expression was significantly reduced in corn seedlings of the Gibberella ear rot resistant genotype CO387 48 h after PBZ treatment. In contrast, the transcript abundance of the maize Zmgc1 gene increased more than 10-fold 8h after the treatment. Therefore, the two genes do not appear to be coordinately regulated by PBZ.

The Multigeneic Rhg1 Locus: A Model For The Effects on Root Development, Nematode Resistance and Recombination Suppression

David A. Lightfoot et al. — Thu, 04 Mar 2010 08:01:37 PST

Soybean (Glycine max L. Merr.) resistance to populations (HgType) of Heterodera glycines I., the soybean cyst nematode (SCN), requires a functional allele at rhg1. An apoptosis-like response in the giant cells formed around the nematode results 24-48 h after feeding commences. This study aimed to identify the role of the three genes within the rhg1 locus, a receptor like kinase (RLK), a laccase and an ion anti-porter. Used were near isogeneic lines (NILs) that contrasted at their rhg1 alleles. Features of the rhg1 locus, the candidate genes and their nascent transcripts and proteins in roots were elucidated. First, evidence for a syntenic gene cluster was found and the effectiveness of SNP probes for distinguishing the homeolog sequence variant on linkage group (Lg) B1 from alleles at the rhg1 locus on Lg G was shown. Analysis of plant s heterozygous at rhg1 showed that the allele for resistance was dominant. The absence of recombination events among the NILs between the RLK and other 2 genes eliminated the possibility of a monogeneic rhg1 locus. Finally, an effect on root development was discovered. A model for multigeneic resistance based on developmental control of root growth including a mechanism for segregation distortion is presented.

In silico comparison of transcript abundances during Arabidopsis thaliana and Glycine max resistance to Fusarium virguliforme

Jiazheng Yuan et al. — Thu, 04 Mar 2010 07:41:37 PST

Background
Sudden death syndrome (SDS) of soybean (Glycine max L. Merr.) is an economically important disease, caused by the semi-biotrophic fungus Fusarium solani f. sp. glycines, recently renamed Fusarium virguliforme (Fv). Due to the complexity and length of the soybean-Fusarium interaction, the molecular mechanisms underlying plant resistance and susceptibility to the pathogen are not fully understood. F. virguliforme has a very wide host range for the ability to cause root rot and a very narrow host range for the ability to cause a leaf scorch. Arabidopsis thaliana is a host for many types of phytopathogens including bacteria, fungi, viruses and nematodes. Deciphering the variations among transcript abundances (TAs) of functional orthologous genes of soybean and A. thaliana involved in the interaction will provide insights into plant resistance to F. viguliforme.

Results
In this study, we reported the analyses of microarrays measuring TA in whole plants after A. thaliana cv 'Columbia' was challenged with fungal pathogen F. virguliforme. Infection caused significant variations in TAs. The total number of increased transcripts was nearly four times more than that of decreased transcripts in abundance. A putative resistance pathway involved in responding to the pathogen infection in A. thaliana was identified and compared to that reported in soybean.

Conclusion
Microarray experiments allow the interrogation of tens of thousands of transcripts simultaneously and thus, the identification of plant pathways is likely to be involved in plant resistance to Fusarial pathogens. Dissection of the set functional orthologous genes between soybean and A. thaliana enabled a broad view of the functional relationships and molecular interactions among plant genes involved in F. virguliforme resistance.

Re-annotation of the physical map of Glycine max for polyploid-like regions by BAC end sequence driven whole genome shotgun read assembly

Navinder Saini et al. — Thu, 04 Mar 2010 07:29:25 PST

Background
Many of the world's most important food crops have either polyploid genomes or homeologous regions derived from segmental shuffling following polyploid formation. The soybean (Glycine max) genome has been shown to be composed of approximately four thousand short interspersed homeologous regions with 1, 2 or 4 copies per haploid genome by RFLP analysis, microsatellite anchors to BACs and by contigs formed from BAC fingerprints. Despite these similar regions,, the genome has been sequenced by whole genome shotgun sequence (WGS). Here the aim was to use BAC end sequences (BES) derived from three minimum tile paths (MTP) to examine the extent and homogeneity of polyploid-like regions within contigs and the extent of correlation between the polyploid-like regions inferred from fingerprinting and the polyploid-like sequences inferred from WGS matches.

Results
Results show that when sequence divergence was 1–10%, the copy number of homeologous regions could be identified from sequence variation in WGS reads overlapping BES. Homeolog sequence variants (HSVs) were single nucleotide polymorphisms (SNPs; 89%) and single nucleotide indels (SNIs 10%). Larger indels were rare but present (1%). Simulations that had predicted fingerprints of homeologous regions could be separated when divergence exceeded 2% were shown to be false. We show that a 5–10% sequence divergence is necessary to separate homeologs by fingerprinting. BES compared to WGS traces showed polyploid-like regions with less than 1% sequence divergence exist at 2.3% of the locations assayed.

Conclusion
The use of HSVs like SNPs and SNIs to characterize BACs wil improve contig building methods. The implications for bioinformatic and functional annotation of polyploid and paleopolyploid genomes show that a combined approach of BAC fingerprint based physical maps, WGS sequence and HSV-based partitioning of BAC clones from homeologous regions to separate contigs will allow reliable de-convolution and positioning of sequence scaffolds (see BES_scaffolds section of SoyGD). This approach will assist genome annotation for paleopolyploid and true polyploid genomes such as soybean and many important cereal and fruit crops.

Soybean Genomics: Developments through the Use of Cultivar “Forrest”

David A. Lightfoot — Thu, 04 Mar 2010 07:19:36 PST

Legume crops are particularly important due to their ability to support symbiotic nitrogen fixation, a key to sustainable crop production and reduced carbon emissions. Soybean (Glycine max) has a special position as a major source of increased protein and oil production in the common grass-legume rotation. The cultivar “Forrest” has saved US growers billions of dollars in crop losses due to resistances programmed into the genome. Moreover, since Forrest grows well in the north-south transition zone, breeders have used this cultivar as a bridge between the southern and northern US gene pools. Investment in Forrest genomics resulted in the development of the following research tools: (i) a genetic map, (ii) three RIL populations (96>n>975), (iii) ~200 NILs, (iv) 115,220 BACs and BIBACs, (v) a physical map, (vi) 4 different minimum tiling path (MTP) sets, (vii) 25,123 BAC end sequences (BESs) that encompass 18.5 Mbp spaced out from the MTPs, and 2 000 microsatellite markers within them (viii) a map of 2,408 regions each found at a single position in the genome and 2104 regions found in 2 or 4 similar copies at different genomic locations (each of >150 kbp), (ix) a map of homoeologous regions among both sets of regions, (x) a set of transcript abundance measurements that address biotic stress resistance, (xi) methods for transformation, (xii) methods for RNAi, (xiii) a TILLING resource for directed mutant isolation, and (xiv) analyses of conserved synteny with other sequenced genomes. The SoyGD portal provides access to the data. To date these resources assisted in the genomic analysis of soybean nodulation and disease resistance. This review summarizes the resources and their uses.

Identification and Use of Actinomycetes for Enhanced Nodulation of Soybean Co-Inoculated with Bradyrhizobium japonicum

A. K. Gregor et al. — Tue, 24 Feb 2009 14:59:02 PST

The utilization of actinomycetes as potential soybean (Glycine max (L.)) co-inoculants was evaluated. Soil samples from Carbondale and Belleville, Ill., were used to inoculate pre-germinated soybean plants to determine antibiotic sensitivity in the native Bradyrhizobium japonicum population. Sensitivity was in the order kanamycin > tetracycline > oxytetracycline > rifampicin > neomycin. Antagonism by five actinomycete cultures toward seven test strains of B. japonicum was also assessed. The ranking average inhibition (across all seven B. japonicum strains) by these actino mycetes was Streptomyces kanamyceticus = Streptomyces coeruleoprunus > Streptomyces rimosus > Streptomyces sp. > Amy colatopsis mediterranei. Ten antibiotic combinations were used to isolate antibiotic-resistant mutants of B. japonicum I-110 and 3I1B-110 via successive cycles of mutation. Eighty-one antibiotic-resistant strains were isolated and tested for symbiotic competency; nine of which were selected for further characterization in a greenhouse pot study. Few differences in nodule number were caused by these treatments. Nodule occupancy varied from 0% to 18.3% when antibiotic-resistant strains of B. japonicum were used as the sole inoculants. However, when three mutant strains of B. japonicum were co-inoculated with S. kanamyceticus, significant increases in nodule occupancy (up to 55%) occurred. Increases in shoot nitrogen composition (27.1%–40.9%) were also caused by co-inoculation with S. kanamyceticus.