The Multigeneic _Rhg1_ Locus: A Model For The Effects on Root Development, Nematode Resistance and Recombination Suppression.

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

Comparative study of physiological and pathological autoimmune models

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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

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 \u27Forrest\u27 that were different among susceptible cultivars \u27Asgrow 3244\u27 and \u27Williams 82\u27 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 rhg

Resistance to Soybean Cyst Nematode: Rhg1

The genes underlying rhg1 lie at a sometimes dominant sometimes co-dominant locus, necessary for resistance to all Hg types of the soybean (Glycine max (L.) Merr.) cyst nematode (Heterodera glycines). Genomic research identified; nucleotide changes within a candidate gene encoding a receptor like kinase (RLK) that were capable of altering root development and thereby part of the resistance to Hg types 0 (race 3); changes in a laccase that are capable of altering cyst development; and genes underlying changes in membrane biology. This set of three genes are subject to co-selection with a modifier locus on another linkage block. Root development is slowed in the resistant seedling and results in end of season yield loss when SCN is not present. However, in the presence of SCN resistant seedling roots grow just as vigorously as the now slower growing parasitized susceptible roots and therefore show little loss to SCN parasitism. In some genotypes but not others the RLK can act alone to confer resistance. Functional paralogs of the three gene cluster have been found on other linkage groups including A1, B1, G, and O and these can be functional in different sources of resistance like G. soja, PI 437654 and PI438489B. At rhg1 the allele differences change the structure, interacting partners and activity of the LRR protein and the laccase. The changes between the alleles result in about 30 other proteins (judged by 2 D gels), 112 metabolites (by FTICRMS) and 8 metabolites (by GCMS) to increase in abundance in roots during SCN infection in the resistant NILs. Understanding the basis of root stunting by resistance alleles will be used to improve methods for developing new nematode resistant soybean cultivars that do not suffer from the yield suppression and low seed germination rates of existing cultivars

The receptor like kinase at Rhg1-a/Rfs2 caused pleiotropic resistance to sudden death syndrome and soybean cyst nematode as a transgene by altering signaling responses

Background: Soybean (Glycine max (L. Merr.)) resistance to any population of Heterodera glycines (I.), or Fusarium virguliforme (Akoi, O’Donnell, Homma & Lattanzi) required a functional allele at Rhg1/Rfs2. H. glycines, the soybean cyst nematode (SCN) was an ancient, endemic, pest of soybean whereas F. virguliforme causal agent of sudden death syndrome (SDS), was a recent, regional, pest. This study examined the role of a receptor like kinase (RLK) GmRLK18-1 (gene model Glyma_18_02680 at 1,071 kbp on chromosome 18 of the genome sequence) within the Rhg1/Rfs2 locus in causing resistance to SCN and SDS. Results: A BAC (B73p06) encompassing the Rhg1/Rfs2 locus was sequenced from a resistant cultivar and compared to the sequences of two susceptible cultivars from which 800 SNPs were found. Sequence alignments inferred that the resistance allele was an introgressed region of about 59 kbp at the center of which the GmRLK18-1 was the most polymorphic gene and encoded protein. Analyses were made of plants that were either heterozygous at, or transgenic (and so hemizygous at a new location) with, the resistance allele of GmRLK18-1. Those plants infested with either H. glycines or F. virguliforme showed that the allele for resistance was dominant. In the absence of Rhg4 the GmRLK18-1 was sufficient to confer nearly complete resistance to both root and leaf symptoms of SDS caused by F. virguliforme and provided partial resistance to three different populations of nematodes (mature female cysts were reduced by 30–50%). In the presence of Rhg4 the plants with the transgene were nearly classed as fully resistant to SCN (females reduced to 11% of the susceptible control) as well as SDS. A reduction in the rate of early seedling root development was also shown to be caused by the resistance allele of the GmRLK18-1. Field trials of transgenic plants showed an increase in foliar susceptibility to insect herbivory. Conclusions: The inference that soybean has adapted part of an existing pathogen recognition and defense cascade (H.glycines; SCN and insect herbivory) to a new pathogen (F. virguliforme; SDS) has broad implications for crop improvement. Stable resistance to many pathogens might be achieved by manipulation the genes encoding a small number of pathogen recognition proteins

QTLs de resistência ao nematoide do cisto da soja, raças 3, 9 e 14 na cultivar Hartwig

The objective of this work was to identify major and minor-effect quantitative trait loci (QTL) for resistance to races 3, 9, and 14 of soybean cyst nematode (SCN) in Hartwig cultivar; to map new resistance QTLs for these races; and to check for the existence of epistatic interactions between QTLs. Cultivar Hartwig is an important resistance source to SCN. Recombinant inbred lines (RIL) obtained from a cross between 'Hartwig' (resistant) and Y23 (susceptible) were evaluated regarding resistance to the three races. New genomic regions for resistance to SCN were identified by microsatellites. Four QTLs, which explained between 12 and 34% of phenotypic variance, were detected for resistance to race 3 in linkage groups (LG) A2, G, J, and M. The QTL in LG G is also important for resistance to race 9. Epistatic interactions were detected between loci, which indicate resistance to races 9 and 14. There are high and low-effect resistance QTLs to SCN.O objetivo deste trabalho foi identificar locos associados a características quantitativas (QTL) de efeito maior e menor para a resistência às raças 3, 9 e 14 do nematoide do cisto da soja (NCS) na cultivar Hartwig, mapear novos QTLs de resistência para estas raças e verificar a existência de interações epistáticas entre QTLs. A cultivar Hartwig é uma importante fonte de resistência ao NCS. Linhagens endogâmicas recombinantes (LER) obtidas do cruzamento entre 'Hartwig' (resistente) e Y23 (suscetível) foram avaliadas quanto à resistência às três raças. Novas regiões genômicas de resistência ao NCS foram identificadas por microssatélites. Quatro QTLs, que explicaram entre 12 e 34% da variância fenotípica, foram detectados para a resistência à raça 3 nos grupos de ligação (GL) A2, G, J e M. O QTL no GL G também é importante para a resistência à raça 9. Interações epistáticas foram detectadas entre loci, o que indica resistência às raças 9 e 14. Há QTLs de maior e menor efeito para a resistência ao NCS.Fil: Ferreira, Marcia Flores da Silva. Universidade Federal do Espírito Santo; BrasilFil: Cervigni, Gerardo Domingo Lucio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad Nacional de Rosario; ArgentinaFil: Ferreira, Adésio. Universidade Federal do Espírito Santo; BrasilFil: Schuster, Iván. Cooperativa Central de Pesquisa Agrícola; BrasilFil: Santana, Fernanda Abreu. Universidade Federal de Viçosa; BrasilFil: Pereira, Waldir Dias. Embrapa Soja; BrasilFil: Barros, Everaldo Gonçalves de. Universidade Federal de Viçosa; BrasilFil: Moreira, Maurilio Alves. Universidade Federal de Viçosa; Brasi

Using A Minimum Tile Path For Plant Transformations Encompassing the Entire Soybean Genome

Genomes like Glycine max (soybean) that have been highly conserved following increases in ploidy represent a frontier for genome analysis. Many soybean QTL analyzed to date have been composed of gene clusters each with contributing a portion of the trait rather than alleles of single genes. At the Soybean Genome Database (SoyGD) http://soybeangenome.siu.edu the genome browser that integrates and served the publicly available soybean physical map, BAC fingerprint database and genetic map associated genomic data shows a minimum tile of transformation ready BIBAC-like clones in pCLD04541 (pV41; oriV; tra; bom). Sequence resources made available through the DOE genome sequencing project have allowed the minimum tile to be revised and new functional analyses to be made. There are 3,840 MTP clones that appeared to encompass 90% of the genome (see http://soybeangenome.siu.edu/cgi-bin/gbrowse/BES_scaffolds). The BIBAC-like clones (tetR) from E. coli DH10 B were transferred en masse to Agrobacterium tumefaciens by triparental matings with EHA105 (rifR) mediated by pRK2013 (oriP) in DH10B (kanR) in 384 well plates. Although not necessary the extra helper plasmid boosted efficiency 10 fold. Individual A. tumefaciens rifampicin and tetracyclin resistant strains were used for transformation of Arabidopsis thaliana flowers in 384 well arrays. Initially kanamycin selection was used to isolate transgenic plants. Because the BACs were already tetR the recA mutants of A. tumefaciens could not be used (Tn3 insertions). Consequent to this and partial transconjugation events only some inserts are transferred completely while other transformed lines contain a substitution series of deleted inserts anchored on the Ti-left border (LB). These are maintained as kanR mixtures of seed. Phenotypes found for lines transgenic for particular BACs that were repeated include seed composition (protein, oil), development (growth, senescence) and disease resistance (suddean death syndrome (SDS) and soybean cyst nematode (SCN)

New Approaches to Selecting Resistance or Tolerance to SDS and Fusarium Root Rot

Fusarial rots are a significant problem worldwide affecting roots (and sometimes fruits) of most major crops including soybean, maize and wheat. Cultivar variation in partial resistance or tolerance is widespread and significant. Different cultivars of the soybean [Glycine max (L.) Merr.] have both resistance/tolerance to the leaf scorch known as Sudden Death Syndrome (SDS) and to the infection and root rot by the causal organism, Fusarium virguliforme (ex. F. solani f. sp glycines) hence the syndrome is composed of two diseases (1-3). Thirteen loci have been identified from analysis of 7 different crosses (2). Using new strains and new methods resistance loci in ‘Hartwig’ and ‘Forrest’, resistant cultivars clearly showed two loci underlie root resistance (lower LG G and D2) and four to eleven loci underlie leaf scorch resistance, depending on the cross made(eg, C2, F, I and upper G in ExF). Transcript abundance analysis of roots in response to F. virguliforme shows an orthologous set of transcripts accumulate during infection of resistant soybean cultivars and Arabidopsis thaliana that include the pathways leading to phenylpropanoid metabolism and its control, guanyl cylase a common second messenger and several transcription factors. Guanyl cyclase is also implicated in resistance in maize. In root disease resistance the genes implicated were known to be stress related. Therefore, A. thaliana is partially resistant and can be used to test both transgenes and mutants in candidate genes. Trangenics show fine maps to BACs have isolated some genes. For example, by fine mapping in NILs candidate genes underlying the controlling loci programming root resistance was a multi-stress resistance protein (lower G; Rfs1). For leaf scorch (Rfs4) an ascorbate peroxidase (C2) has been targeted. Also, Rfs2, a receptor like kinase (G) has been used to generate stable transgenic soybeans. Identification of the genes and loci conferring SDS resistance has provided options to breed improved cultivars with resistance to SDS