Margaret H. MacDonald

Identification of molecular markers in soybean comparing RFLP, RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.

Population-specific gene expression in the plant pathogenic nematode Heterodera glycines exists prior to infection and during the onset of a resistant or susceptible reaction in the roots of the Glycine max genotype Peking.

BackgroundA single Glycine max (soybean) genotype (Peking) reacts differently to two different populations of Heterodera glycines (soybean cyst nematode) within the first twelve hours of infection during resistant (R) and susceptible (S) reactions. This suggested that H. glycines has population-specific gene expression signatures. A microarray analysis of 7539 probe sets representing 7431 transcripts on the Affymetrix® soybean GeneChip® were used to identify population-specific gene expression signatures in pre-infective second stage larva (pi-L2) prior to their infection of Peking. Other analyses focused on the i nfective L2 at 12h ours post infection (i-L212h), and the i nfective sedentary stages at 3d ays post infection (i-L23d) and 8d ays post infection (i-L2/L38d).ResultsDifferential expression and false discovery rate (FDR) analyses comparing populations of pi-L2 (i.e., incompatible population, NL1-RHg to compatible population, TN8) identified 71 genes that were induced in NL1-RHg as compared to TN8. These genes included putative gland protein G23G12, putative esophageal gland protein Hgg-20 and arginine kinase. The comparative analysis of pi-L2 identified 44 genes that were suppressed in NL1-RHg as compared to TN8. These genes included a different Hgg-20 gene, an EXPB1 protein and a cuticular collagen. By 12 h, there were 7 induced genes and 0 suppressed genes in NL1-RHg. By 3d, there were 9 induced and 10 suppressed genes in NL1-RHg. Substantial changes in gene expression became evident subsequently. At 8d there were 13 induced genes in NL1-RHg. This included putative gland protein G20E03, ubiquitin extension protein, putative gland protein G30C02 and β-1,4 endoglucanase. However, 1668 genes were found to be suppressed in NL1-RHg. These genes included steroid alpha reductase, serine proteinase and a collagen protein.ConclusionThese analyses identify a genetic expression signature for these two populations both prior to and subsequently as they undergo an R or S reaction. The identification of genes like steroid alpha reductase and serine proteinase that are involved in feeding and nutritional uptake as being highly suppressed during the R response at 8d may indicate genes that the plant is targeting. The analyses also identified numerous putative parasitism genes that are differentially expressed. The 1668 genes that are suppressed in NL1-RHg, and hence induced in TN8 may represent genes that are important during the parasitic stages of H. glycines development. The potential for different arrays of putative parasitism genes to be expressed in different nematode populations may indicate how H. glycines evolve mechanisms to overcome resistance.

Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes

BackgroundThe gene encoding PAD4 (PHYTOALEXIN-DEFICIENT4) is required in Arabidopsis for expression of several genes involved in the defense response to Pseudomonas syringae pv. maculicola. AtPAD4 (Arabidopsis thaliana PAD4) encodes a lipase-like protein that plays a regulatory role mediating salicylic acid signaling.ResultsWe expressed the gene encoding AtPAD4 in soybean roots of composite plants to test the ability of AtPAD4 to deter plant parasitic nematode development. The transformed roots were challenged with two different plant parasitic nematode genera represented by soybean cyst nematode (SCN; Heterodera glycines) and root-knot nematode (RKN; Meloidogyne incognita). Expression of AtPAD4 in soybean roots decreased the number of mature SCN females 35 days after inoculation by 68 percent. Similarly, soybean roots expressing AtPAD4 exhibited 77 percent fewer galls when challenged with RKN.ConclusionsOur experiments show that AtPAD4 can be used in an economically important crop, soybean, to provide a measure of resistance to two different genera of nematodes.

Molecular markers residing close to the Rhg4 locus conferring resistance to soybean cyst nematode race 3 on linkage group A of soybean

Abstract The restriction fragment length polymorphism (RFLP) clone pBLT65 is a 450-nt soybean cDNA encoding a portion of the bifunctional enzyme aspartokinase-homoserine dehydrogenase (AK-HSDH). pBLT65 maps within 3.5 cM of the i locus, conferring a pigmented seed coat, on linkage group A; hence, it is closely linked to the Rhg4 locus conferring resistance to race 3 of the soybean cyst nematode. From this useful RFLP we developed a PCR reaction yielding polymorphic bands for use in marker-assisted breeding programs to select progeny containing the Rhg4 allele. The polymorphic bands were sequenced to determine the cause of the polymorphisms. Using primers 548 and 563, PCR amplification of DNA from the soybean cultivar Peking (Rhg4) yielded three DNA fragments, 1a (1160 bp), 1b (1146 bp) and 3 (996 bp). Amplification of DNA from the cultivar Kent (rhg4) yielded DNA fragments 2 (1020 bp), 3 (996 bp) and 4 (960 bp). Fragments 1a, 1b, 2 and 4 were also polymorphic between the soybean lines PI 290136 and BARC-2(Rj4). A segregating population of 80 F2 and F3 plants derived from the cross PI 290136×BARC-2 (Rj4) was used to confirm the map position of the PCR polymorphisms near the i locus, and hence the Rhg4 locus on linkage group A. The nucleotide sequences of fragments 1b, 3 and 4 were determined. Large and small deletions in the intronic region were responsible for the size differences of the different fragments, whereas the exon was well conserved.

Arabidopsis genes, AtNPR1, AtTGA2 and AtPR-5, confer partial resistance to soybean cyst nematode (Heterodera glycines) when overexpressed in transgenic soybean roots

BackgroundExtensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) and derivatives are critical to the defense response against necrotrophic pathogens. Several reports demonstrate that SA limits nematode reproduction.ResultsHere we translate knowledge gained from studies using Arabidopsis to soybean. The ability of thirty-one Arabidopsis genes encoding important components of SA and JA synthesis and signaling in conferring resistance to soybean cyst nematode (SCN: Heterodera glycines) are investigated. We demonstrate that overexpression of three of thirty-one Arabidoposis genes in transgenic soybean roots of composite plants decreased the number of cysts formed by SCN to less than 50% of those found on control roots, namely AtNPR1(33%), AtTGA2 (38%), and AtPR-5 (38%). Three additional Arabidopsis genes decreased the number of SCN cysts by 40% or more: AtACBP3 (53% of the control value), AtACD2 (55%), and AtCM-3 (57%). Other genes having less or no effect included AtEDS5 (77%), AtNDR1 (82%), AtEDS1 (107%), and AtPR-1 (80%), as compared to control. Overexpression of AtDND1 greatly increased susceptibility as indicated by a large increase in the number of SCN cysts (175% of control).ConclusionsKnowledge of the pathogen defense system gained from studies of the model system, Arabidopsis, can be directly translated to soybean through direct overexpression of Arabidopsis genes. When the genes, AtNPR1, AtGA2, and AtPR-5, encoding specific components involved in SA regulation, synthesis, and signaling, are overexpressed in soybean roots, resistance to SCN is enhanced. This demonstrates functional compatibility of some Arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes.

Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines)

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process.

MiniMax, a new diminutive Glycine max genotype with a rapid life cycle, embryogenic potential and transformation capabilities

We developed Glycine max cv MiniMax (PI643148) that has a rapid life cycle, short stature and characteristic simple sequence repeat (SSR) markers that could make it useful for mutant screening, functional genomics, genetic mapping and other studies involving soybeans. We demonstrate that MiniMax is able to make somatic embryos (SEs) that rapidly develop into plantlets. Thus, the rapid cycling habit carries over into aspects of plant regeneration. Chimaeras (having transformed roots with untransformed aerial stocks) have been produced rapidly under non-axenic conditions using Agrobacterium rhizogenes-mediated transformation. Part of these experiments involved the engineering an enhanced green fluorescent protein (eGFP) reporter cassette outside the multi-cloning site of a plant expression vector, permitting non-invasive visual screening of the transformed roots. The rapid cycling growth habit of MiniMax, its ability to efficiently generate SEs and ability to be transformed should prove useful for basic aspects of G. max molecular and genetic research.

A decline in transcript abundance for Heterodera glycines homologs of Caenorhabditis elegans uncoordinated genes accompanies its sedentary parasitic phase

BackgroundHeterodera glycines (soybean cyst nematode [SCN]), the major pathogen of Glycine max (soybean), undergoes muscle degradation (sarcopenia) as it becomes sedentary inside the root. Many genes encoding muscular and neuromuscular components belong to the uncoordinated (unc) family of genes originally identified in Caenorhabditis elegans. Previously, we reported a substantial decrease in transcript abundance for Hg-unc-87, the H. glycines homolog of unc-87 (calponin) during the adult sedentary phase of SCN. These observations implied that changes in the expression of specific muscle genes occurred during sarcopenia.ResultsWe developed a bioinformatics database that compares expressed sequence tag (est) and genomic data of C.elegans and H.glycines (CeHg database). We identify H. glycines homologs of C. elegans unc genes whose protein products are involved in muscle composition and regulation. RT-PCR reveals the transcript abundance of H. glycines unc homologs at mobile and sedentary stages of its lifecycle. A prominent reduction in transcript abundance occurs in samples from sedentary nematodes for homologs of actin, unc-60B (cofilin), unc-89, unc-15 (paromyosin), unc-27 (troponin I), unc-54 (myosin), and the potassium channel unc-110 (twk-18). Less reduction is observed for the focal adhesion complex gene Hg-unc-97.ConclusionThe CeHg bioinformatics database is shown to be useful in identifying homologs of genes whose protein products perform roles in specific aspects of H. glycines muscle biology. Our bioinformatics comparison of C. elegans and H. glycines genomic data and our Hg-unc-87 expression experiments demonstrate that the transcript abundance of specific H. glycines homologs of muscle gene decreases as the nematode becomes sedentary inside the root during its parasitic feeding stages.

Two‐dimensional proteome reference maps for the soybean cyst nematode Heterodera glycines

2‐DE reference maps of Heterodera glycines were constructed. After in‐gel digestion with trypsin, 803 spots representing 426 proteins were subsequently identified by LC‐MS/MS. Proteins with annotated function were further categorized by Gene Ontology. The results showed that proteins involved in metabolic, developmental and biological regulation processes were the most abundant.

The pJan25 vector series: An enhancement of the Gateway-compatible vector pGWB533 for broader promoter testing applications

Agrobacterium-mediated transformation of plants has enhanced our ability to progress more rapidly in plant genetic engineering. Development of binary vectors for Agrobacterium has played a major role in advancing plant biology. Here, we report new features added to the Gateway-compatible vector pGWB533 for promoter testing with the reporter gene encoding β-glucuronidase (GUS). The original vector contains the spectinomycin/streptomycin adenylyltransferase (aadA) gene for bacterial selection and the hygromycin phosphotransferase gene (hpt) for transformed plant selection. However, some bacterial strains used to transform plants, such as Agrobacterium rhizogenes strain K599, have elevated tolerance to spectinomycin and streptomycin, thus making bacterial selection of pGWB533 inefficient. Although pGWB533 confers chemical selection for transgenic plants using hygromycin resistance, the plasmid has no visual marker that enables visual selection of transformed plants or transgenic tissue. In this regard, adding a gene to constitutively express green fluorescent protein (eGFP) makes it easier to visually select the transformed tissue and trim out the non-transformed. In this report we describe a series of vectors, pJan25S (NCBI: KC416200), pJan25T (NCBI: KC416201) and pJan25X (NCBI: KC416202), that are enhancements of pGWB533 for promoter testing. All three vectors contain the gene encoding eGFP as a visual marker for transformed tissue. However, in pJan25S and pJan25T, eGFP is controlled by the rolD promoter for root-specific expression, while in pJan25X it is controlled by the CaMV35S promoter for constitutive expression in all plant tissues. Spectinomycin and streptomycin resistance remains in pJan25S for bacterial selection; however, pJan25T and pJan25X contain the gene encoding tetracycline resistance (tet) for bacterial selection. These changes resulted in enhanced vectors with better visual and chemical selection that should have broad application in promoter studies.